Methods and Compositions for Diagnosing Disease

ABSTRACT

The present invention relates to methods and compositions for diagnosing a disease or disorder in a subject by introducing into cells of the subject a diagnostic gene switch construct and monitoring expression of a reporter gene. The invention further relates to methods and compositions for monitoring the progression of a disease or disorder or the effectiveness of a treatment for a disease or disorder.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the sequence listing text file (File Name: SequenceListing.ST25.txt; Size: 107 KB bytes; and Date of Creation: Aug. 22,2008) filed herewith with the application is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions for diagnosinga disease or disorder in a subject by introducing into cells of thesubject a diagnostic gene switch construct and monitoring expression ofa reporter gene. The invention further relates to methods andcompositions for monitoring the progression of a disease or disorder ormonitoring the effectiveness or toxicity of a treatment for a disease ordisorder.

2. Background Art

Diagnostic tests for the presence of a disease in a subject have longbeen in existence, but researchers are constantly searching for improvedtests exhibiting increased sensitivity (allowing earlier detection) andspecificity (eliminating false positives and false negatives). Otherdesired characteristics for diagnostic tests include ease of use, rapidresults, and the ability to constantly monitor progression of a diseaseor the effectiveness of ongoing treatment.

Thus, there is a need in the art for new diagnostic methods andcompositions that provide these desired characteristics.

SUMMARY OF THE INVENTION

The present invention is based on a combination of the specificity andsensitivity provided by the use of disease specific promoters to detecta disease coupled with the regulatory control of a ligand-dependent geneswitch system to provide diagnostic and monitoring methods. The presentinvention relates to methods and compositions for diagnosing a diseaseor disorder in a subject. The invention further relates to methods andcompositions for monitoring the progression of a disease or disorder ina subject or monitoring the effectiveness or toxicity of an administeredtreatment for a disease or disorder in a subject.

One embodiment of the invention comprises methods of diagnosing adisease or disorder in a subject, comprising:

-   (a) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (2) administering ligand to said modified cells; and-   (3) detecting reporter gene expression;

wherein expression of the reporter gene indicates that said subject hassaid disease or disorder.

In one embodiment, the diagnostic methods are carried out ex vivo incells that have been isolated from said subject.

In one embodiment, the diagnostic methods are carried out by introducingthe compositions of the invention into cells that have been isolatedfrom said subject to produce modified cells, and the modified cells arere-introduced into said subject.

In one embodiment, the diagnostic methods are carried out in vivo.

In a further embodiment, the diagnostic methods are carried out usingnon-autologous cells, e.g., cells that are allogeneic or xenogeneic tothe subject, and the modified non-autologous cells are introduced intothe subject. In one embodiment, the non-autologous cells are surroundedby a barrier (e.g., encapsulated) prior to being introduced into thesubject.

In one aspect of the invention, the gene switch is an ecdysone receptor(EcR)-based gene switch.

In one embodiment, the gene switch comprises a first transcriptionfactor sequence under the control of a first diagnostic switch promoterand a second transcription factor sequence under the control of a seconddiagnostic switch promoter,

wherein the proteins encoded by said first transcription factor sequenceand said second transcription factor sequence interact to form a proteincomplex which functions as a ligand-dependent transcription factor.

In another aspect of the invention, said first transcription factorsequence encodes a protein comprising a heterodimer partner and atransactivation domain and said second transcription factor sequenceencodes a protein comprising a DNA binding domain and a ligand-bindingdomain.

An additional embodiment of the invention relates to methods ofmonitoring the progression of a disease or disorder in a subject,comprising:

-   (a) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said disease or disorder in said subject.

A further embodiment of the invention relates to methods of monitoringthe effectiveness of a treatment for a disease or disorder in a subject,comprising:

-   (a) administering said treatment to said subject;-   (b) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the effectiveness of said treatment.

Another embodiment of the invention relates to methods of monitoring thepotential toxicity of an administered treatment for a disease ordisorder in a subject, comprising:

-   (a) administering said treatment to said subject;-   (b) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, linked to a diagnostic switch promoter,    wherein the activity of the promoter is modulated by factors found    in cells that are being exposed to toxic conditions, and (2) a    polynucleotide encoding a reporter gene linked to a promoter which    is activated by said ligand-dependent transcription factor, to    produce modified cells;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the toxicity of said treatment.

Another embodiment of the invention relates to methods of monitoring thelevel of a factor that is being administered to a subject for treatmentfor a disease or disorder, comprising:

-   (a) administering said treatment to said subject;-   (b) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, linked to a diagnostic switch promoter,    wherein the activity of the promoter is modulated by said factor    that is being administered for treatment, and (2) a polynucleotide    encoding a reporter gene linked to a promoter which is activated by    said ligand-dependent transcription factor, to produce modified    cells;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression;

wherein the level of expression of said reporter gene indicates thelevel of the factor being administered for treatment.

In a further embodiment, each of the methods may be carried out usingnon-autologous cells, e.g., cells that are allogeneic or xenogeneic tothe subject, and the modified non-autologous cells are administered tothe subject. In one embodiment, the modified non-autologous cells aresurrounded by a barrier (e.g., encapsulated) prior to being introducedinto the subject.

One embodiment of the invention comprises methods of detectingtransplant rejection in a subject that has received an organ or tissuetransplant, comprising:

-   (a) introducing into cells of said organ or tissue transplant (1) a    polynucleotide encoding a gene switch, said gene switch comprising    at least one transcription factor sequence, wherein said at least    one transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    transplant rejection, and (2) a polynucleotide encoding a reporter    gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression;

wherein expression of the reporter gene indicates that transplantrejection has been detected.

An additional embodiment of the invention relates to methods ofmonitoring the progression of transplant rejection in a subject that hasreceived an organ or tissue transplant, comprising:

-   (a) introducing into cells of said organ or tissue transplant (1) a    polynucleotide encoding a gene switch, said gene switch comprising    at least one transcription factor sequence, wherein said at least    one transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    transplant rejection, and (2) a polynucleotide encoding a reporter    gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said transplant rejection in said subject.

In a further embodiment, the methods of detecting or monitoringtransplant rejection may be carried out by introducing thepolynucleotides of the invention into non-autologous cells, e.g., cellsthat are allogeneic or xenogeneic to the organ or tissue beingtransplanted, and the modified non-autologous cells are introduced tothe organ or tissue prior to transplantation. In one embodiment, themodified non-autologous cells are surrounded by a barrier (e.g.,encapsulated) prior to being introduced into the organ or tissue.

In the methods described above, in one embodiment, the polynucleotideencoding the gene switch and the polynucleotide encoding the reportergene linked to a promoter are part of one larger polynucleotide, e.g., avector. In another embodiment, the polynucleotide encoding the geneswitch and the polynucleotide encoding the reporter gene linked to apromoter are separate polynucleotides.

The invention further relates to diagnostic gene switch constructs thatare useful in the disclosed methods.

The invention additionally relates to vectors comprising the diagnosticgene switch constructs of the invention.

The invention also relates to kits for carrying out the methods of theinvention, comprising, e.g., gene switch constructs, vectors, ligands,etc. In one embodiment, the kits may comprise cells (e.g., autologous ornon-autologous cells) that may comprise the polynucleotides of theinvention. The non-autologous cells may be surrounded by a barrier(e.g., encapsulated).

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows an embodiment of the diagnostic gene switch of theinvention in which two transcription factor sequences encoding twoseparate portions of a ligand-dependent transcription factor are underthe control of different promoters. “Dx-Switch Components” represents agene switch; “AD” represents a transactivation domain; “DBD” representsa DNA binding domain; “LBD” represents a ligand binding domain;“StandardDx-Reporter” represents a reporter gene; and “P1” and “P2”represent two different disease- or disorder-responsive promoters. In analternative embodiment of FIG. 1, “P1” is a constitutive promoter; and“P2” and “P3” are different disease- or disorder-responsive promoters.

FIG. 2 shows an embodiment of the diagnostic gene switch of theinvention in which two transcription factor sequences encoding twoseparate portions of a ligand-dependent transcription factor are underthe control of different promoters. “Dx-Switch Components” represents agene switch; “AD” represents a transactivation domain; “DBD-A”represents a first DNA binding domain; “DBD-B” represents a second DNAbinding domain; “LBD” represents a ligand binding domain;“StandardDx-Reporter-A” represents a first reporter gene;“StandardDx-Reporter-B” represents a second reporter gene; and “P1,”“P2,” and “P3” represent three different disease- or disorder-responsivepromoters. In an alternative embodiment of FIG. 2, “P1” is aconstitutive promoter; and “P2” and “P3” are different disease- ordisorder-responsive promoters.

FIG. 3 shows an embodiment of the diagnostic gene switch of theinvention in which two transcription factor sequences encoding twoseparate portions of a ligand-dependent transcription factor are underthe control of different diagnostic switch promoters. “Dx-SwitchComponents” represents a gene switch; “AD” represents a transactivationdomain; “DBD” represents a DNA binding domain; “LBD” represents a ligandbinding domain; “StandardDx-Reporter” represents a reporter gene; and“P1,” “P2,” “P3,” and “P4” represent four different disease- ordisorder-responsive promoters.

FIG. 4 shows an embodiment of the diagnostic gene switch of theinvention in which two transcription factor sequences encoding twoseparate portions of a ligand-dependent transcription factor are underthe control of different promoters and a control reporter gene ispresent. “Dx-Switch Components” represents a gene switch; “AD”represents a transactivation domain; “DBD-A” represents a first DNAbinding domain; “DBD-B” represents a second DNA binding domain; “LBD”represents a ligand binding domain; “StandardDx-Reporter-A” represents afirst reporter gene; “Control-Reporter-B” represents a second reportergene; and “P1” and “P2” represent two different disease- ordisorder-responsive promoters; and “P3” and “P4” represent two differentcontrol promoters. In an alternative embodiment of FIG. 4, “P3” and “P4”are constitutive promoters.

FIG. 5 shows an embodiment of a single promoter shuttle vector (SEQ IDNo.: 5), which includes the IL-24/mda-7 promoter. Adenovirus producedusing this vector is used to transduce cells isolated from lymphaticsamples.

FIG. 6 shows an embodiment of a dual promoter vector (SEQ ID NO.: 6),which includes TRPM4 and TRGC1/TARP promoters. This DNA vector is usedto transduce a prostate biopsy using non-viral transduction systems.

FIG. 7 shows an embodiment of a single promoter vector (SEQ ID NO.: 7),which includes the ADAM-17 promoter and the CD95-ADAM8 dual reporter(SEQ ID NO.: 10).

FIG. 8 shows an embodiment of a dual promoter vector (SEQ ID NO.: 8),which includes the CXCL9 and SEMA7A promoters and the CD40-CD3 dualreporter (SEQ ID NO.: 12).

FIG. 9 shows an embodiment of a single promoter vector (SEQ ID NO.: 9),which includes the ADAM-17 promoter and the alkaline phosphatase-cterminal CD40 reporter (SEQ ID NO.: 14).

FIG. 10 shows embodiments of the serum-based reporters that are designedto exhibit no immunogenic profile when expressed within the human body.These reporters are made up of human based amino acid sequences that arepresent either on the cell surface or within the serum naturally. Hence,these reporters are not immunogenic nor are they subject to immuneattack when expressed in the human body. In one embodiment, serum-basedreporters are dual epitope reporter, for e.g., CD95-ADAM8 reporter (SEQID NOs.: 10-11), CD40-CD3 reporter (SEQ ID NOs.: 12-13), CD28-CD3reporter (SEQ ID. NOs.: 16-17) and CD28-CD40 reporter (SEQ ID NOs.:18-19) that allows ELISA based capture and detection. The designsutilize a signal peptide (Signal P) for transport into the secretorypathway, followed by epitopes from cell surface antigens with linkers(L). In alternative embodiments, different combinations of linkers andepitopes are used for each design. In another embodiment, theserum-based reporter is an alkaline phosphatase reporter, for e.g.,alkaline phosphatase-c terminal CD40 reporter (SEQ ID NOs.: 14-15), thatallows immunocapture followed by enzymatic detection of reporteractivity. Alkaline phosphatase reporters utilize the tissue non-specificalkaline phosphatase for an enzymatic reporter that can be secreted. Anepitope from a cell surface antigen is included at the carboxy terminusfor immunocapture prior to measurement of alkaline phosphatase activity.In additional embodiments of FIG. 10, additional alkaline phosphatasereporters are: alkaline phosphatase—amiono terminal CD40 reporter (SEQID NOs.: 20-21) and alkaline phosphatase—c terminal CD28 reporter (SEQID. NOs.: 22-23).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods and compositions for using a geneswitch for the diagnosis of diseases or disorders in a subject. Theinvention further relates to methods and compositions for monitoring theprogression of diseases or disorders or the treatment thereof in asubject. The methods of the invention can be carried out either ex vivo(by introducing the gene switch into isolated cells of a subject) or invivo (by introducing the gene switch into isolated cells of a subjectand reintroducing the cells to the subject or by introducing the geneswitch directly into cells of the subject). In another embodiment, thecells harboring the gene switch may be non-autologous cells (e.g.,allogeneic or xenogeneic cells). The non-autologous cells may besurrounded by a barrier that prevents the non-autologous cells fromraising an immune response after introduction and/or prevents thenon-autologous cells from escaping from the site of introduction. Themethods of the invention involve the use of a gene switch in whichexpression of a ligand-dependent transcription factor is under thecontrol of one or more diagnostic switch promoters. The methods andcompositions described herein provide a highly sensitive and highlyspecific diagnostic technique in which the timing of the diagnostic stepis controlled by administration of ligand to cells comprising the geneswitch, permitting optimal detection of the presence of a disease ordisorder as well as continuous or intermittent monitoring of theprogression of a disease or disorder or the effectiveness or toxicity ofa treatment.

The following definitions are provided and should be helpful inunderstanding the scope and practice of the present invention.

The term “isolated” for the purposes of the present invention designatesa biological material (cell, nucleic acid or protein) that has beenremoved from its original environment (the environment in which it isnaturally present). For example, a polynucleotide present in the naturalstate in a plant or an animal is not isolated, however the samepolynucleotide separated from the adjacent nucleic acids in which it isnaturally present, is considered “isolated.”

The term “purified,” as applied to biological materials does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

“Nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” and“polynucleotide” are used interchangeably and refer to the phosphateester polymeric form of ribonucleosides (adenosine, guanosine, uridineor cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), orany phosphoester analogs thereof, such as phosphorothioates andthioesters, in either single stranded form, or a double-stranded helix.Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. Theterm nucleic acid molecule, and in particular DNA or RNA molecule,refers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear or circularDNA molecules (e.g., restriction fragments), plasmids, supercoiled DNAand chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the non-transcribed strand of DNA (i.e., thestrand having a sequence homologous to the mRNA). A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation. DNA includes, but is not limited to, cDNA, genomic DNA,plasmid DNA, synthetic DNA, and semi-synthetic DNA.

The term “fragment,” as applied to polynucleotide sequences, refers to anucleotide sequence of reduced length relative to the reference nucleicacid and comprising, over the common portion, a nucleotide sequenceidentical to the reference nucleic acid. Such a nucleic acid fragmentaccording to the invention may be, where appropriate, included in alarger polynucleotide of which it is a constituent. Such fragmentscomprise, or alternatively consist of, oligonucleotides ranging inlength from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25,30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90,100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000,3000, 4000, 5000, or more consecutive nucleotides of a nucleic acidaccording to the invention.

As used herein, an “isolated nucleic acid fragment” refers to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to a polynucleotide comprising nucleotides that encode afunctional molecule, including functional molecules produced bytranscription only (e.g., a bioactive RNA species) or by transcriptionand translation (e.g., a polypeptide). The term “gene” encompasses cDNAand genomic DNA nucleic acids. “Gene” also refers to a nucleic acidfragment that expresses a specific RNA, protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Heterologous DNA” refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. The heterologous DNA may include agene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook et al. in Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor (1989), particularly Chapter 11 and Table 11.1 therein (entirelyincorporated herein by reference). The conditions of temperature andionic strength determine the “stringency” of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS. Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SSC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SSC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the present invention also includes isolatednucleic acid fragments that are complementary to the complete sequencesas disclosed or used herein as well as those substantially similarnucleic acid sequences.

In one embodiment of the invention, polynucleotides are detected byemploying hybridization conditions comprising a hybridization step atT_(m), of 55° C., and utilizing conditions as set forth above. In otherembodiments, the T_(m) is 60° C., 63° C., or 65° C.

Post-hybridization washes also determine stringency conditions. One setof conditions uses a series of washes starting with 6×SSC, 0.5% SDS atroom temperature for 15 minutes (min), then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS is increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In one embodiment of the invention, polynucleotides are detected byemploying hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37° C., and a washing step in 2×SSPEat a temperature of at least 63° C. In another embodiment, thehybridization conditions comprise less than 200 mM salt and at least 37°C. for the hybridization step. In a further embodiment, thehybridization conditions comprise 2×SSPE and 63° C. for both thehybridization and washing steps.

In another embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferably a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; e.g., atleast about 20 nucleotides; e.g., at least 30 nucleotides. Furthermore,the skilled artisan will recognize that the temperature and washsolution salt concentration may be adjusted as necessary according tofactors such as length of the probe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a short nucleicacid that is hybridizable to a genomic DNA molecule, a cDNA molecule, aplasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g.,with ³²P-nucleotides or nucleotides to which a label, such as biotin,has been covalently conjugated. A labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. Oligonucleotides (oneor both of which may be labeled) can be used as PCR primers, either forcloning full length or a fragment of a nucleic acid, for DNA sequencing,or to detect the presence of a nucleic acid. An oligonucleotide can alsobe used to form a triple helix with a DNA molecule. Generally,oligonucleotides are prepared synthetically, preferably on a nucleicacid synthesizer. Accordingly, oligonucleotides can be prepared withnon-naturally occurring phosphoester analog bonds, such as thioesterbonds, etc.

A “primer” refers to an oligonucleotide that hybridizes to a targetnucleic acid sequence to create a double stranded nucleic acid regionthat can serve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction orfor DNA sequencing.

“Polymerase chain reaction” is abbreviated PCR and refers to an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand refers to an in vitro method for enzymatically producing a targetcDNA molecule or molecules from an RNA molecule or molecules, followedby enzymatic amplification of a specific nucleic acid sequence orsequences within the target cDNA molecule or molecules as describedabove. RT-PCR also provides a means to detect the presence of the targetmolecule and, under quantitative or semi-quantitative conditions, todetermine the relative amount of that target molecule within thestarting pool of nucleic acids.

A DNA “coding sequence” refers to a double-stranded DNA sequence thatencodes a polypeptide and can be transcribed and translated into apolypeptide in a cell in vitro or in vivo when placed under the controlof suitable regulatory sequences. “Suitable regulatory sequences” refersto nucleotide sequences located upstream (5′ non-coding sequences),within, or downstream (3′ non-coding sequences) of a coding sequence,and which influence the transcription, RNA processing or stability, ortranslation of the associated coding sequence. Regulatory sequences mayinclude promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures. The boundaries of the codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, prokaryotic sequences, cDNAfrom mRNA, genomic DNA sequences, and even synthetic DNA sequences. Ifthe coding sequence is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

“Open reading frame” is abbreviated ORF and refers to a length ofnucleic acid sequence, either DNA, cDNA or RNA, that comprises atranslation start signal or initiation codon, such as an ATG or AUG, anda termination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (← →) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→ ←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→ →)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to a reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” are usedinterchangeably and refer to an enzyme that binds and cuts within aspecific nucleotide sequence within double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” refers to any genetic element(e.g., plasmid, phage, cosmid, chromosome, virus) that functions as anautonomous unit of DNA replication in vivo, i.e., capable of replicationunder its own control. The term “vector” includes both viral andnonviral vehicles for introducing the nucleic acid into a cell in vitro,ex vivo or in vivo. A large number of vectors known in the art may beused to manipulate nucleic acids, incorporate response elements andpromoters into genes, etc. Possible vectors include, for example,plasmids or modified viruses including, for example bacteriophages suchas lambda derivatives, or plasmids such as pBR322 or pUC plasmidderivatives, or the Bluescript vector. Another example of vectors thatare useful in the present invention is the UltraVector™ ProductionSystem (Intrexon Corp., Blacksburg, Va.) as described in WO 2007/038276,incorporated herein by reference. For example, the insertion of the DNAfragments corresponding to response elements and promoters into asuitable vector can be accomplished by ligating the appropriate DNAfragments into a chosen vector that has complementary cohesive termini.Alternatively, the ends of the DNA molecules may be enzymaticallymodified or any site may be produced by ligating nucleotide sequences(linkers) into the DNA termini. Such vectors may be engineered tocontain selectable marker genes that provide for the selection of cellsthat have incorporated the marker into the cellular genome. Such markersallow identification and/or selection of host cells that incorporate andexpress the proteins encoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include, but are notlimited to, retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid, preferably DNA, that replicates sequentially and whichcomprises an origin of replication, such as a plasmid, phage or cosmid,to which another nucleic acid segment may be attached so as to bringabout the replication of the attached segment. Cloning vectors may becapable of replication in one cell type and expression in another(“shuttle vector”). Cloning vectors may comprise one or more sequencesthat can be used for selection of cells comprising the vector and/or oneor more multiple cloning sites for insertion of sequences of interest.

The term “expression vector” refers to a vector, plasmid or vehicledesigned to enable the expression of an inserted nucleic acid sequencefollowing transformation into the host. The cloned gene, i.e., theinserted nucleic acid sequence, is usually placed under the control ofcontrol elements such as a promoter, a minimal promoter, an enhancer, orthe like. Initiation control regions or promoters, which are useful todrive expression of a nucleic acid in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving expression of these genes can be used in an expressionvector, including but not limited to, viral promoters, bacterialpromoters, animal promoters, mammalian promoters, synthetic promoters,constitutive promoters, tissue specific promoters, pathogenesis ordisease related promoters, developmental specific promoters, induciblepromoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10,ADH1, PGK, PHOS, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkalinephosphatase promoters (useful for expression in Saccharomyces); AOX1promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet,tip, lP_(L), lP_(R), T7, tac, and trc promoters (useful for expressionin Escherichia coli); light regulated-, seed specific-, pollenspecific-, ovary specific-, cauliflower mosaic virus 35S, CMV 35Sminimal, cassava vein mosaic virus (CsVMV), chlorophyll a/b bindingprotein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, rootspecific, chitinase, stress inducible, rice tungro bacilliform virus,plant super-promoter, potato leucine aminopeptidase, nitrate reductase,mannopine synthase, nopaline synthase, ubiquitin, zein protein, andanthocyanin promoters (useful for expression in plant cells); animal andmammalian promoters known in the art including, but are not limited to,the SV40 early (SV40e) promoter region, the promoter contained in the 3′long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters ofthe E1A or major late promoter (MLP) genes of adenoviruses (Ad), thecytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV)thymidine kinase (TK) promoter, a baculovirus IE1 promoter, anelongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase(PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, theregulatory sequences of the mouse metallothionein-L promoter andtranscriptional control regions, the ubiquitous promoters (HPRT,vimentin, α-actin, tubulin and the like), the promoters of theintermediate filaments (desmin, neurofilaments, keratin, GFAP, and thelike), the promoters of therapeutic genes (of the MDR, CFTR or factorVIII type, and the like), pathogenesis or disease related-promoters, andpromoters that exhibit tissue specificity and have been utilized intransgenic animals, such as the elastase I gene control region which isactive in pancreatic acinar cells; insulin gene control region active inpancreatic beta cells, immunoglobulin gene control region active inlymphoid cells, mouse mammary tumor virus control region active intesticular, breast, lymphoid and mast cells; albumin gene, Apo AI andApo AII control regions active in liver, alpha-fetoprotein gene controlregion active in liver, alpha 1-antitrypsin gene control region activein the liver, beta-globin gene control region active in myeloid cells,myelin basic protein gene control region active in oligodendrocyte cellsin the brain, myosin light chain-2 gene control region active inskeletal muscle, and gonadotropic releasing hormone gene control regionactive in the hypothalamus, pyruvate kinase promoter, villin promoter,promoter of the fatty acid binding intestinal protein, promoter of thesmooth muscle cell α-actin, and the like. In addition, these expressionsequences may be modified by addition of enhancer or regulatorysequences and the like.

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963(1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al.,Canadian Patent Application No. 2,012,311).

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Feigner et al., Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey etal., Proc. Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al.,Science 259:1745 (1993)). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Feigner et al., Science337:387 (1989)). Particularly useful lipid compounds and compositionsfor transfer of nucleic acids are described in WO95/18863, WO96/17823and U.S. Pat. No. 5,459,127. The use of lipofection to introduceexogenous genes into the specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly preferred in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (Mackey et al. 1988, supra). Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther. 3:147 (1992); and Wu et al., J. Biol. Chem.262:4429 (1987)).

The term “transfection” refers to the uptake of exogenous orheterologous RNA or DNA by a cell. A cell has been “transfected” byexogenous or heterologous RNA or DNA when such RNA or DNA has beenintroduced inside the cell. A cell has been “transformed” by exogenousor heterologous RNA or DNA when the transfected RNA or DNA effects aphenotypic change. The transforming RNA or DNA can be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” refers to an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” refers to a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the transcription of a nucleicacid. Promoters may be derived in their entirety from a native gene, orbe composed of different elements derived from different promoters foundin nature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmental orphysiological conditions. Promoters that cause a gene to be expressed inmost cell types at most times are commonly referred to as “constitutivepromoters.” Promoters that cause a gene to be expressed in a specificcell type are commonly referred to as “cell-specific promoters” or“tissue-specific promoters.” Promoters that cause a gene to be expressedat a specific stage of development or cell differentiation are commonlyreferred to as “developmentally-specific promoters” or “celldifferentiation-specific promoters.” Promoters that are induced andcause a gene to be expressed following exposure or treatment of the cellwith an agent, biological molecule, chemical, ligand, light, or the likethat induces the promoter are commonly referred to as “induciblepromoters” or “regulatable promoters.” It is further recognized thatsince in most cases the exact boundaries of regulatory sequences havenot been completely defined, DNA fragments of different lengths may haveidentical promoter activity.

The promoter sequence is typically bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding oftranscription factors that recruit RNA polymerase-mediatedtranscription.

“Diagnostic switch promoter” refers to a promoter the activity of whichis modulated by a factor in a manner that can be used as a diagnostic inthe present invention. The term encompasses promoters that increase ordecrease expression of a coding sequence during a disease or disorder asa change in promoter activity in either direction will be diagnostic.The term includes, without limitation, disease-specific promoters,promoters responsive to particular physiological or pathologicalconditions, and promoters responsive to specific biological molecules.Diagnostic switch promoters can comprise the sequence of naturallyoccurring promoters, modified sequences derived from naturally occurringpromoters, or synthetic sequences (e.g., insertion of a response elementinto a promoter sequence to alter the responsiveness of the promoter).

A “coding sequence” is a DNA sequence that encodes a polypeptide or aRNA (e.g., a functional RNA).

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into RNA. If the coding sequence is aprotein coding sequence, the primary RNA transcript is then furtherprocessed (e.g., trans-RNA spliced (if the coding sequence containsintrons) and polyadenylated), exported to the cytoplasm, and translatedinto the protein encoded by the coding sequence. Non-protein-codingbioactive RNA species (including, but not limited to RNAi or microRNAs)can be functional in the nucleus as a primary transcript, a splicedtranscript (with or without polyadenylation), and/or an excised intron;or can exert bioactivity in extra-nuclear cellular regions as any RNAform that is exported from the nucleus.

“Transcriptional and translational control sequences” refer to DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

The term “response element” refers to one or more cis-acting DNAelements which confer responsiveness on a promoter mediated throughinteraction with the DNA-binding domains of a transcription factor. ThisDNA element may be either palindromic (perfect or imperfect) in itssequence or composed of sequence motifs or half sites separated by avariable number of nucleotides. The half sites can be similar oridentical and arranged as either direct or inverted repeats or as asingle half site or multimers of adjacent half sites in tandem. Theresponse element may comprise a minimal promoter isolated from differentorganisms depending upon the nature of the cell or organism into whichthe response element will be incorporated. The DNA binding domain of thetranscription factor binds, in the presence or absence of a ligand, tothe DNA sequence of a response element to initiate or suppresstranscription of downstream gene(s) under the regulation of thisresponse element. Examples of DNA sequences for response elements of thenatural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 1)(see Cherbas et. al., Genes Dev. 5:120 (1991)); AGGTCAN_((n))AGGTCA,where N_((n)) can be one or more spacer nucleotides (SEQ ID NO: 2) (seeD'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995)); andGGGTTGAATGAATTT (SEQ ID NO: 3) (see Antoniewski et al., Mol. Cell Biol.14:4465 (1994)).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein refers to the production of RNA(e.g., sense RNA, antisense RNA, microRNA, messenger RNA, heterologousnuclear RNA, ribosomal RNA, small interfering RNA, ribozymes, etc.) bytranscription of a nucleic acid or polynucleotide. Expression may alsoinclude translation of mRNA into a protein or polypeptide.

The terms “cassette,” “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and aligand-dependent transcription factor-based system which, in thepresence of one or more ligands, modulates the expression of a gene intowhich the response element and promoter are incorporated.

The term “ecdysone-based,” with respect to a gene switch, refers to agene switch comprising at least a functional part of a naturallyoccurring or synthetic ecdysone receptor ligand binding domain and whichregulates gene expression in response to a ligand that binds to theecdysone receptor ligand binding domain.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The polynucleotides or vectors according to the invention may furthercomprise at least one promoter suitable for driving expression of a genein a host cell.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In a one embodiment of theinvention, the termination control region may be comprised or be derivedfrom a synthetic sequence, synthetic polyadenylation signal, an SV40late polyadenylation signal, an SV40 polyadenylation signal, a bovinegrowth hormone (BGH) polyadenylation signal, viral terminator sequences,or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” refers to a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” refers to a regulatoryregion that is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (tnRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

“Polypeptide,” “peptide” and “protein” are used interchangeably andrefer to a polymeric compound comprised of covalently linked amino acidresidues.

An “isolated polypeptide,” “isolated peptide” or “isolated protein”refer to a polypeptide or protein that is substantially free of thosecompounds that are normally associated therewith in its natural state(e.g., other proteins or polypeptides, nucleic acids, carbohydrates,lipids). “Isolated” is not meant to exclude artificial or syntheticmixtures with other compounds, or the presence of impurities which donot interfere with biological activity, and which may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into a pharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one wild-type or naturally occurring amino acid with a differentamino acid relative to the wild-type or naturally occurring polypeptide.A substitution mutant polypeptide may comprise only one wild-type ornaturally occurring amino acid substitution and may be referred to as a“point mutant” or a “single point mutant” polypeptide. Alternatively, asubstitution mutant polypeptide may comprise a substitution of two ormore wild-type or naturally occurring amino acids with two or more aminoacids relative to the wild-type or naturally occurring polypeptide.According to the invention, a Group H nuclear receptor ligand bindingdomain polypeptide comprising a substitution mutation comprises asubstitution of at least one wild-type or naturally occurring amino acidwith a different amino acid relative to the wild-type or naturallyoccurring Group H nuclear receptor ligand binding domain polypeptide.

When the substitution mutant polypeptide comprises a substitution of twoor more wild-type or naturally occurring amino acids, this substitutionmay comprise either an equivalent number of wild-type or naturallyoccurring amino acids deleted for the substitution, i.e., 2 wild-type ornaturally occurring amino acids replaced with 2 non-wild-type ornon-naturally occurring amino acids, or a non-equivalent number ofwild-type amino acids deleted for the substitution, i.e., 2 wild-typeamino acids replaced with 1 non-wild-type amino acid (asubstitution+deletion mutation), or 2 wild-type amino acids replacedwith 3 non-wild-type amino acids (a substitution+insertion mutation).

Substitution mutants may be described using an abbreviated nomenclaturesystem to indicate the amino acid residue and number replaced within thereference polypeptide sequence and the new substituted amino acidresidue. For example, a substitution mutant in which the twentieth(20^(th)) amino acid residue of a polypeptide is substituted may beabbreviated as “x20z”, wherein “x” is the amino acid to be replaced,“20” is the amino acid residue position or number within thepolypeptide, and “z” is the new substituted amino acid. Therefore, asubstitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala”indicates that the mutant comprises an alanine residue (commonlyabbreviated in the art as “A” or “Ala”) in place of the glutamic acid(commonly abbreviated in the art as “E” or “Glu”) at position 20 of thepolypeptide.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978); Zolleret al., DNA 3:479 (1984); Oliphant et al., Gene 44:177 (1986);Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986)), use ofTAB® linkers (Pharmacia), restriction endonuclease digestion/fragmentdeletion and substitution, PCR-mediated/oligonucleotide-directedmutagenesis, and the like. PCR-based techniques are preferred forsite-directed mutagenesis (see Higuchi, 1989, “Using PCR to EngineerDNA”, in PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

The term “fragment,” as applied to a polypeptide, refers to apolypeptide whose amino acid sequence is shorter than that of thereference polypeptide and which comprises, over the entire portion withthese reference polypeptides, an identical amino acid sequence. Suchfragments may, where appropriate, be included in a larger polypeptide ofwhich they are a part. Such fragments of a polypeptide according to theinvention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200,240, or 300 or more amino acids.

A “variant” of a polypeptide or protein refers to any analogue,fragment, derivative, or mutant which is derived from a polypeptide orprotein and which retains at least one biological property of thepolypeptide or protein. Different variants of the polypeptide or proteinmay exist in nature. These variants may be allelic variationscharacterized by differences in the nucleotide sequences of thestructural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canproduce variants having single or multiple amino acid substitutions,deletions, additions, or replacements. These variants may include, interalia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids, (b)variants in which one or more amino acids are added to the polypeptideor protein, (c) variants in which one or more of the amino acidsincludes a substituent group, and (d) variants in which the polypeptideor protein is fused with another polypeptide such as serum albumin. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art. Inone embodiment, a variant polypeptide comprises at least about 14 aminoacids.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667 (1987)). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the present application, theterm “homologous,” when modified with an adverb such as “highly,” mayrefer to sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., Cell 50:667 (1987)). In oneembodiment, two DNA sequences are “substantially homologous” or“substantially similar” when at least about 50% (e.g., at least about75%, 90%, or 95%) of the nucleotides match over the defined length ofthe DNA sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data banks, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart (see e.g., Sambrook et al., 1989, supra).

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate alteration of gene expression by antisense or co-suppressiontechnology. “Substantially similar” also refers to modifications of thenucleic acid fragments of the present invention such as deletion orinsertion of one or more nucleotide bases that do not substantiallyaffect the functional properties of the resulting transcript. It istherefore understood that the invention encompasses more than thespecific exemplary sequences. Each of the proposed modifications is wellwithin the routine skill in the art, as is determination of retention ofbiological activity of the encoded products.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the present invention are those nucleic acid fragmentswhose DNA sequences are at least about 70%, 80%, 90% or 95% identical tothe DNA sequence of the nucleic acid fragments reported herein.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al., J. Mol. Biol. 215:403 (1993)); available atncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing sequence analysis software such as the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences may be performed using the Clustalmethod of alignment (Higgins et al., CABIOS. 5:151 (1989)) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method may beselected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwareincludes, but is not limited to, the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)),and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA).Within the context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Chemically synthesized,” as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentially of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism, e.g., at least5-fold, 10-fold, 100-fold, or 500-fold greater. Ideally, modulation ofeach of the given systems by its respective ligand at a chosenconcentration results in a measurable change in the magnitude ofexpression of the gene of that system and no measurable change inexpression of all other systems operable in the cell, tissue, ororganism. In such cases the multiple inducible gene regulation system issaid to be “fully orthogonal.” The present invention is useful to searchfor orthogonal ligands and orthogonal receptor-based gene expressionsystems such as those described in US 2002/0110861 A1, which isincorporated herein by reference in its entirety.

The term “exogenous gene” means a gene foreign to the subject, that is,a gene which is introduced into the subject through a transformationprocess, an unmutated version of an endogenous mutated gene or a mutatedversion of an endogenous unmutated gene. The method of transformation isnot critical to this invention and may be any method suitable for thesubject known to those in the art. Exogenous genes can be either naturalor synthetic genes and therapeutic genes which are introduced into thesubject in the form of DNA or RNA which may function through a DNAintermediate such as by reverse transcriptase. Such genes can beintroduced into target cells, directly introduced into the subject, orindirectly introduced by the transfer of transformed cells into thesubject. The term “therapeutic gene” means a gene which imparts abeneficial function to the host cell in which such gene is expressed.

The term “ecdysone receptor complex” generally refers to a heterodimericprotein complex having at least two members of the nuclear receptorfamily, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins(see Yao et al., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)).The functional EcR complex may also include additional protein(s) suchas immunophilins. Additional members of the nuclear receptor family ofproteins, known as transcriptional factors (such as DHR38, betaFTZ-1 orother insect homologs), may also be ligand dependent or independentpartners for EcR and/or USP. The EcR complex can also be a heterodimerof EcR protein and the vertebrate homolog of ultraspiracle protein,retinoic acid-X-receptor (“RXR”) protein. The term EcR complex alsoencompasses homodimer complexes of the EcR protein or USP.

An EcR complex can be activated by an active ecdysteroid ornon-steroidal ligand bound to one of the proteins of the complex,inclusive of EcR, but not excluding other proteins of the complex. Asused herein, the term “ligand,” as applied to EcR-based gene switches,describes small and soluble molecules having the capability ofactivating a gene switch to stimulate expression of a polypeptideencoded therein. Examples of ligands include, without limitation, anecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A,muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs ofretinoic acid, N,N′-diacylhydrazines such as those disclosed in U.S.Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S.Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolinesas described in U.S. Published Application No. 2004/0171651;dibenzoylalkyl cyanohydrazines such as those disclosed in EuropeanApplication No. 461,809; N-alkyl-N,N′-diaroylhydrazines such as thosedisclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazinessuch as those disclosed in European Application No. 234,994;N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.No. 4,985,461; amidoketones such as those described in U.S. PublishedApplication No. 2004/0049037; each of which is incorporated herein byreference and other similar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide,oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonateesters, juvenile hormone III, and the like. Examples of diacylhydrazineligands useful in the present invention include RG-115819(3,5-Dimethyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-methyl-3-methoxy-benzoyl)-hydrazide),RG-115830 (3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide), andRG-115932 ((R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide). SeeU.S. application Ser. No. 12/155,111.

The EcR complex includes proteins which are members of the nuclearreceptor superfamily wherein all members are characterized by thepresence of an amino-terminal transactivation domain (“TA”), a DNAbinding domain (“DBD”), and a ligand binding domain (“LBD”) separated bya hinge region. Some members of the family may also have anothertransactivation domain on the carboxy-terminal side of the LBD. The DBDis characterized by the presence of two cysteine zinc fingers betweenwhich are two amino acid motifs, the P-box and the D-box, which conferspecificity for ecdysone response elements. These domains may be eithernative, modified, or chimeras of different domains of heterologousreceptor proteins.

The DNA sequences making up the exogenous gene, the response element,and the EcR complex may be incorporated into archaebacteria, procaryoticcells such as Escherichia coli, Bacillus subtilis, or otherenterobacteria, or eucaryotic cells such as plant or animal cells.However, because many of the proteins expressed by the gene areprocessed incorrectly in bacteria, eucaryotic cells are preferred. Thecells may be in the form of single cells or multicellular organisms. Thenucleotide sequences for the exogenous gene, the response element, andthe receptor complex can also be incorporated as RNA molecules,preferably in the form of functional viral RNAs such as tobacco mosaicvirus. Of the eucaryotic cells, vertebrate cells are preferred becausethey naturally lack the molecules which confer responses to the ligandsof this invention for the EcR. As a result, they are “substantiallyinsensitive” to the ligands of this invention. Thus, the ligands usefulin this invention will have negligible physiological or other effects ontransformed cells, or the whole organism. Therefore, cells can grow andexpress the desired product, substantially unaffected by the presence ofthe ligand itself.

The term “subject” means an intact insect, plant or animal. It is alsoanticipated that the ligands will work equally well when the subject isa fungus or yeast. When the subject is an intact animal, preferably theanimal is a vertebrate, most preferably a mammal.

EcR ligands, when used with the EcR complex which in turn is bound tothe response element linked to an exogenous gene (e.g., a reportergene), provide the means for external temporal regulation of expressionof the exogenous gene. The order in which the various components bind toeach other, that is, ligand to receptor complex and receptor complex toresponse element, is not critical. Typically, modulation of expressionof the exogenous gene is in response to the binding of the EcR complexto a specific control, or regulatory, DNA element. The EcR protein, likeother members of the nuclear receptor family, possesses at least threedomains, a transactivation domain, a DNA binding domain, and a ligandbinding domain. This receptor, like a subset of the nuclear receptorfamily, also possesses less well-defined regions responsible forheterodimerization properties. Binding of the ligand to the ligandbinding domain of EcR protein, after heterodimerization with USP or RXRprotein, enables the DNA binding domains of the heterodimeric proteinsto bind to the response element in an activated form, thus resulting inexpression or suppression of the exogenous gene. This mechanism does notexclude the potential for ligand binding to either EcR or USP, and theresulting formation of active homodimer complexes (e.g. EcR+EcR orUSP+USP). In one embodiment, one or more of the receptor domains can bevaried producing a chimeric gene switch. Typically, one or more of thethree domains may be chosen from a source different than the source ofthe other domains so that the chimeric receptor is optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988) orLexA protein from E. coli (see Brent et al., Cell 43:729 (1985)) toaccommodate chimeric EcR complexes. Another advantage of chimericsystems is that they allow choice of a promoter used to drive theexogenous gene according to a desired end result. Such double controlcan be particularly important in areas of gene therapy, especially whencytotoxic proteins are produced, because both the timing of expressionas well as the cells wherein expression occurs can be controlled. Whenexogenous genes, operatively linked to a suitable promoter, areintroduced into the cells of the subject, expression of the exogenousgenes is controlled by the presence of the ligand of this invention.Promoters may be constitutively or inducibly regulated or may betissue-specific (that is, expressed only in a particular type of cell)or specific to certain developmental stages of the organism.

Numerous genomic and cDNA nucleic acid sequences coding for a variety ofpolypeptides, such as transcription factors and reporter genes, are wellknown in the art. Those skilled in the art have access to nucleic acidsequence information for virtually all known genes and can either obtainthe nucleic acid molecule directly from a public depository, theinstitution that published the sequence, or employ routine methods toprepare the molecule.

For in vivo use, the ligands described herein may be taken up inpharmaceutically acceptable carriers, such as, for example, solutions,suspensions, tablets, capsules, ointments, elixirs, and injectablecompositions. Pharmaceutical compositions may contain from 0.01% to 99%by weight of the ligand. Compositions may be either in single ormultiple dose forms. The amount of ligand in any particularpharmaceutical composition will depend upon the effective dose, that is,the dose required to elicit the desired gene expression or suppression.

Suitable routes of administering the pharmaceutical preparations includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural) and by naso-gastric tube. It willbe understood by those skilled in the art that the preferred route ofadministration will depend upon the condition being diagnosed and mayvary with factors such as the condition of the recipient.

One embodiment of the invention comprises methods of diagnosing adisease or disorder in a subject, comprising:

-   (a) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression;

wherein expression of the reporter gene indicates that said subject hassaid disease, disorder, or condition.

In one embodiment, the diagnostic methods are carried out ex vivo incells that have been isolated from said subject.

In one embodiment, the diagnostic methods are carried out by introducingthe compositions of the invention into cells that have been isolatedfrom said subject to produce modified cells, and the modified cells arere-introduced into said subject.

In one embodiment, the diagnostic methods are carried out in vivo.

In a different embodiment, the diagnostic methods may be carried outusing non-autologous cells, e.g., cells that are allogeneic orxenogeneic to the subject, instead of autologous cells from the subject.The polynucleotides may be introduced into the non-autologous cells exvivo to produce modified cells and the modified cells may then beintroduced into the subject. The non-autologous cells may be any cellsthat are viable after transplantation into a subject, including, withoutlimitation, stem cells (such as embryonic stem cells or hematopoieticstem cells) and fibroblasts.

One embodiment of the invention relates to methods of diagnosing adisease or disorder in a subject, comprising:

-   (a) introducing into non-autologous cells (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) introducing said modified cells into said subject;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression;

wherein expression of said reporter gene indicates that said subject hassaid disease or disorder.

In one aspect of this embodiment, the modified cells are surrounded by abarrier (e.g., encapsulated) prior to being introduced into the subject.The encapsulated cells will function as an implantable biosensor. In oneembodiment, encapsulation of cells and methods for making them areprovided, which provide improved structural characteristics and immuneprotection. Such encapsulated cells will withstand mechanical, chemicalor immune destruction within the host, and will additionally provide forfree permeability to nutrients, ions, oxygen, and other materials neededto both maintain the cell and support normal metabolic functions. In oneembodiment, the encapsulated cells are impermeable to bacteria,lymphocytes, and large proteins of the type responsible forimmunochemical reactions. In one embodiment, the barrier will alsofunction to prevent the non-autologous cells from escaping from the siteof introduction, e.g., rogue cells that might cause harm to the subjectif allowed to circulate. In one embodiment, the barrier is a selectivelypermeable barrier, e.g., a barrier that is permeable to small moleculessuch as hormones and small peptides but impermeable to largerpolypeptides such as antibodies. For example, the barrier may beimpermeable to molecules with a molecular weight greater than about100,000, about 50,000, about 25,000, about 10,000, about 5,000 or about1,000 daltons.

Two encapsulation methods, microencapsulation and macroencapsulation,are known in the art. Typically, microencapsulated cells are sequesteredin a small spherical container, whereas macroencapsulated cells areentrapped in a larger non-spherical membrane. For encapsulation, livingcells and other sensitive materials are treated under sufficiently mildconditions allowing the cells or biomaterial to remain substantiallyunaffected by the encapsulation process, yet permitting the formation ofa capsule of sufficient strength to exist over long periods of time.

In one embodiment, the cells are encapsulated within a biocompatiblesemi-permeable membrane. The term “biocompatible” as used herein referscollectively to both the intact capsule and its contents. Specifically,it refers to the capability of the implanted intact encapsulated cell toavoid detrimental effects of the body's various protective systems, suchas immune system or foreign body fibrotic response, and remainfunctional for a significant period of time.

The capsules of the present invention are especially useful for theadministration of cells by injection, implantation or transplantation toa subject. Living cells can be encapsulated in a variety of gels, toform implantable devices, e.g., microbeads or microspheres to physicallyisolate the cells once implanted into a host. To prevent entry ofsmaller molecular weight substances such as antibodies and complement(with a molecular weight of about 150 kDa) into these mircoparticles,they can be coated with a material such as poly-L-lysine, chitosan, orPAN-PVC, which provides an outer shell with a controlled pore size orthey can be treated by e.g., cross-linking, to control their internalporosity. Additional examples of useful materials include conventionalbiocompatible materials made up of natural or synthetic polymers orco-polymers, such as alginate, poly-L-lysine-alginate, collagen,gelatin, laminin, methyl methacrylate, hydroxyethyl methacrylate,MATRIGEL, VIRTOGEN, polyvinylalcohol, agarose, polyethylene glycol,hydrogels, polylactic acid, polyglycolic acid,poly(lactide-co-glycolide), polyhydroxybutyrate-polyhydroxyvalerate,copolymer, poly(lactide-co-caprolactone), polyesteramides,polyorthoesters, poly 13-hydroxybutyric acid, polyanhydrides,polyethylene terephthalate, polyetrafluoroethylene, polyacrylates(including acrylic copolymers), polyvinylidenes, polyvinyl chloridecopolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates,cellulose nitrates, polysulfones (including polyether sulfones),polyphosphazenes, polyacrylonitriles, and poly(acrylonitrile/covinylchloride).

In one embodiment, the cells are isolated and suspended in liquid mediumand then encapsulated by a supporting matrix, e.g., a hydrogel matrix toform a microbead. This microbead may serve as a core of an implantabledevice. The core will maintain a proper cell distribution, providestrength, and enhance cell viability, longevity, and function. The corewill also contribute to immunoisolation. The core will also protect theinternal particle from direct cell-cell interactions that can elicit anundesirable host response.

The barrier may contain multiple layers, e.g., where each layer serves adifferent purpose (e.g., support, control of permeability). Barriers mayalso comprise contrast agents or other properties that render thebarrier imageable (e.g., by x-ray, sonography, etc.) to ensure properpositioning of the implanted cells. Examples of barrier systems usefulfor cell implantation are described in U.S. Pat. Nos. 7,226,978,RE39,542 (agarose), 6,960,351, 6,916,640, 6,911,227 (polyethyleneglycol), 6,818,018, 6,808,705, 6,783,964, 6,762,959, 6,727,322,6,610,668 (poly-14-N-acetylglucosamine (p-GlcNAc) polysaccharide),6,558,665, RE38,027, 6,495,161, 6,368,612, 6,365,385, 6,337,008,6,306,454 (polyalkylene), 6,303,355, 6,287,558 (gel super matrix),6,281,015, 6,264,941, 6,258,870, 6,180,007, 6,126,936 (polyamine acid),6,123,700, 6,083,523, 6,020,200, 5,916,790, 5,912,005, 5,908,623,5,902,745, 5,858,746, 5,846,530 (polysaccaharides), 5,843,743,5,837,747, 5,837,234, 5,834,274, 5,834,001, 5,801,033, 5,800,829,5,800,828, 5,798,113, 5,788,988, 5,786,216, 5,773,286, 5,759,578,5,700,848, 5,656,481, 5,653,975, 5,648,099, 5,550,178, 4,806,355,4,689,293, 4,680,174, 4,673,566, 4,409,331, 4,352,883, and U.S. PatentApplication Publications 2006/0263405 (alginate/polymer) and2004/0005302 (alignate-poly-L-lysine), each incorporated herein byreferences in its entirety.

In one embodiment, the polynucleotide encoding the gene switch and thepolynucleotide encoding the reporter gene linked to a promoter are partof one larger polynucleotide, e.g., a vector. In another embodiment, thepolynucleotide encoding the gene switch and the polynucleotide encodingthe reporter gene linked to a promoter are separate polynucleotides.

The subject on which the diagnostic methods are carried out may be anysubject for which a diagnosis is desired. For example, the subject maybe one that is exhibiting one or more symptoms of a disease or disorder.The subject may also be one that is predisposed to a disease ordisorder, e.g., due to genetics, family history, or environmentalexposure. The subject may be a member of the general public, e.g., aspart of a screening for the prevalence of a disease or disorder in apopulation.

The disease or disorder to be diagnosed by the methods of the inventionmay be any disease or disorder for which one or more diagnostic switchpromoters are available. Examples of diseases or disorders which may bediagnosed by the methods of the invention include, without limitation,hyperproliferative diseases (e.g., cancer), cardiovascular diseases,neural diseases, autoimmune diseases, graft versus host disease,transplant rejection, bone diseases, gastrointestinal diseases, blooddiseases, metabolic diseases, inflammatory diseases, and infections.

One embodiment of the invention relates to methods of preparing modifiedcells for diagnosing a disease or disorder in a subject, comprisingintroducing into cells of said subject (1) a polynucleotide encoding agene switch, said gene switch comprising at least one transcriptionfactor sequence, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, operably linked to adiagnostic switch promoter, wherein the activity of the promoter ismodulated during said disease or disorder, and (2) a polynucleotideencoding a reporter gene linked to a promoter which is activated by saidligand-dependent transcription factor, to produce modified cells.

Another embodiment of the invention relates to methods of diagnosing adisease or disorder in a subject, comprising:

-   (a) administering ligand to modified cells of said subject; and-   (b) detecting reporter gene expression;

wherein expression of said reporter gene indicates that said subject hassaid disease or disorder, and

wherein said modified cells of said subject comprise (1) apolynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, operably linked to a diagnostic switch promoter, wherein theactivity of the promoter is modulated during said disease or disorder,and (2) a polynucleotide encoding a reporter gene linked to a promoterwhich is activated by said ligand-dependent transcription factor.

The diagnostic switch promoters of the invention may be any promoterthat is useful for diagnosing a specific disease or disorder, monitoringthe progression of a disease, or monitoring the effectiveness ortoxicity of a treatment. Examples include, without limitation, promotersof genes that exhibit increased or decreased expression only during aspecific disease or disorder and promoters of genes that exhibitincreased or decreased expression under specific cell conditions (e.g.,proliferation, apoptosis, change in pH, oxidation state, oxygen level).In some embodiments where the gene switch comprises more than onetranscription factor sequence, the specificity of the diagnostic methodscan be increased by combining a disease- or condition-specific promoterwith a tissue- or cell type-specific promoter to limit the tissues inwhich a diagnostic measurement occurs. Thus, tissue- or celltype-specific promoters are encompassed within the definition ofdiagnostic switch promoter.

As an example of disease-specific promoters, useful promoters fordiagnosing cancer include the promoters of oncogenes. Examples ofclasses of oncogenes include, but are not limited to, growth factors,growth factor receptors, protein kinases, programmed cell deathregulators and transcription factors. Specific examples of oncogenesinclude, but are not limited to, sis, erb B, erb B-2, ras, abl, myc andbcl-2 and TERT. Examples of other cancer-related genes include tumorassociated antigen genes and other genes that are overexpressed inneoplastic cells (e.g., MAGE-1, carcinoembryonic antigen, tyrosinase,prostate specific antigen, prostate specific membrane antigen, p53,MUC-1, MUC-2, MUC-4, HER-2/neu, T/Tn, MART-1, gp100, GM2, Tn, sTn, andThompson-Friedenreich antigen (TF)).

Examples of promoter sequences and other regulatory elements (e.g.,enhancers) that are known in the art and are useful as diagnostic switchpromoters in the present invention are disclosed in the referenceslisted in Tables 1 and 2, along with the disease/disorder (Table 1) ortissue specificity (Table 2) associated with each promoter. The promotersequences disclosed in these references are herein incorporated byreference in their entirety.

TABLE 1 Patent/ Published Application Promoter Sequence Disease/DisorderNo. Her-2/neu (ERBB2/c-erbB-2) cancer 5,518,885 osteocalcin calcifiedtumors 5,772,993 stromelysin-1 cancer 5,824,794 prostate specificantigen prostate cancer 5,919,652 human sodium-iodide symporter thyroidcarcinoma 6,015,376 H19, IF-1, IGF-2 cancer 6,306,833 thymosin β15breast, pancreatic, 6,489,463 prostate cancer T cell factor cancer6,608,037 cartilage-derived retinoic acid- chondrosarcoma, 6,610,509sensitive protein mammary tumor insulin pancreatic cancer 6,716,824PEG-3 cancer 6,737,523 telomerase reverse transcriptase cancer 6,777,203melanoma differentiation associated cancer 6,841,362 gene-7 prostasincancer 6,864,093 telomerase catalytic subunit; cancer 6,936,595 cyclin-Amidkine; c-erbB-2 cancer 7,030,099 prostate-specific membrane antigenprostate cancer 7,037,647 p51 cancer 7,038,028 telomerase RNA cancer7,084,267 prostatic acid phosphatase prostate cancer 7,094,533PCA3_(dd3) prostate cancer 7,138,235 DF3/MUC1 cancer 7,247,297 hex IIcancer 2001/0011128 cyclooxygenase-2 cancer 2002/0107219 super PSAprostate cancer 2003/0078224 skp2 cancer 2003/0109481 PRL-3 metastaticcolon 2004/0126785 cancer CA125/M17S2 ovarian cancer 2004/0126824 IAI.3Bovarian cancer 2005/0031591 CRG-L2 liver cancer 2005/0124068 TRPM4prostate cancer 2006/0188990 RTVP glioma 2006/0216731 TARP prostatecancer, 2007/0032439 breast cancer telomere reverse transcriptase cancer2007/0059287 A4 amyloid protein Alzheimer's 5,151,508 disease amyloidβ-protein precursor Alzheimer's 5,643,726 disease precursor of theAlzheimer's Disease Alzheimer's 5,853,985 A4 amyloid protein diseaseneuropeptide FF CNS disorders 6,320,038 endoplasmic reticulum stressstress 7,049,132 elements urocortin II psycho- 7,087,385 pathologiestyrosine hydroxylase neurological 7,195,910 disorders complement factor3; serum amyloid inflammation 5,851,822 A3 tissue inhibitor ofmetalloproteinase- rheumatism, 5,854,019 3 (TIMP-3) cancer, autoimmunedisease, inflammation p75 tumor necrosis factor receptor autoimmune5,959,094 disease tumor necrosis factor-α inflammation 6,537,784peroxisome proliferator activated inflammation 6,870,044 receptor/IIA-1nonpancreatic secreted phospholipase A2 SOCS-3 growth 2002/0174448disorders, autoimmune disease, inflammation SR-BI lipid disorders5,965,790 Ob obesity 5,698,389 site-1 protease obesity, diabetes7,045,294 TIGR glaucoma 7,138,511 VL30 anoxia 5,681,706 excitatory aminoacid transporter-2 nervous system 2004/0171108 ischemia MDTS9 renalfailure 2006/0014931 LIM, pyrroline 5-carboxylate prostate disorders2006/0134688 reductase, SIM2 Bax apoptosis 5,744,310 fas apoptosis5,888,764 bbc3 apoptosis 7,202,024 PINK-1 PI-3 kinase/Akt 2006/0228776pathway disorders

TABLE 2 Patent/Published Promoter Sequence Tissue SpecificityApplication No. troponin T skeletal muscle 5,266,488 myoD muscle5,352,595 actin muscle 5,374,544 smooth muscle 22α arterial smooth5,837,534 muscle utrophin muscle 5,972,609 myostatin muscle 6,284,882smooth muscle myosin heavy chain smooth muscle 6,780,610 cardiac ankyrinrepeat protein cardiac muscle 7,193,075 MLP muscle 2002/0042057smoothelin smooth muscle 2003/0157494 MYBPC3 cardiomyocytes 2004/0175699Tα1 α-tubulin neurons 5,661,032 intercellular adhesion molecule-4neurons 5,753,502 (ICAM-4) γ-aminobutyric acid type A hippocampus6,066,726 receptor β1 subunit neuronal nicotinic acetylcholine neurons6,177,242 receptor β2-subunit presenilin-1 neurons 6,255,473calcium-calmodulin-dependent forebrain 6,509,190 kinase IIα CRF_(2α)receptor brain 7,071,323 nerve growth factor neurons 2003/159159 GLP-2receptor gut, brain 2002/0045173 type I transglutaminase keratinocytes5,643,746 K14 keratinocytes 6,596,515 stearoyl-CoA desaturase skin2002/0151018 megsin renal cells 6,790,617 prolactin pituitary 5,082,779GDF-9 ovary, testes, 7,227,013 hypothalamus, pituitary, placenta PSP94prostate 2003/0110522 NRL; NGAL mammary gland 5,773,290 long whey acidicprotein mammary gland 5,831,141 mammary associated amyloid A mammaryductal 2005/0107315 epithelial cells endothelin-1 endothelial cells5,288,846 serglycin hematopoietic 5,340,739 cells platelet-endothelialcell adhesion platelets, 5,668,012 molecule-1 (PECAM-1) leukocytes,endothelial cells Tie receptor tyrosine kinase endothelial cells,5,877,020 bone marrow KDR/flk-1 endothelial cells 5,888,765 endoglinendothelial cells 6,103,527 CCR5 myeloid and 6,383,746 lymphoid cellsCD11d myeloid cells 6,881,834 platelet glycoprotein IIb hematopoietic6,884,616 cells preproendothelin-1 endothelial cells 7,067,649interleukin-18 binding protein mononuclear cells 2006/0239984 CD34hematopoietic 5,556,954 stem cells Tec tyrosine kinase hematopoietic6,225,459 stem cells, liver AC133 stem cells 2005/0125849

Other genes that exhibit changes in expression levels during specificdiseases or disorders and therefore are useful in the present inventioninclude, without limitation, the genes (along with the associateddisease/disorder) listed in Table 3.

TABLE 3 Patent/Published Gene Disease/Disorder Application No. MLH1,MSH2, MSH6, PMS1, APC Colorectal cancer 7,148,016 LEF-1 Colon cancer2002/0169300 F₂ receptor Colon cancer 2002/0187502 TGF-β type IIreceptor Colon cancer 2004/0038284 EYA4 Colon cancer 2005/0003463 PCA3Prostate cancer 7,138,235 K2 Prostate cancer 6,303,361 PROST 03 Prostatecancer metastases 2002/0009455 PCAM-1 Prostate cancer 2002/0042062PCADM-1 Prostate cancer 2003/0100033 PCA3_(dd3) Prostate cancer2003/0165850 PCAV Prostate cancer 2006/0275747 PAcP Androgen-insensitive2006/0294615 prostate cancer SEQ ID NO: 1 of the patent Liver cancer5,866,329 5,866,329, incorporated by reference herein SEQ ID NOS: 1, 3of the U.S. patent Hepatocellular cancer 2002/0115094 applicationpublication 2002/0115094, incorporated by reference herein SEQ ID NO: 1of the patent U.S. Hepatocellular carcinoma 2005/0037372 applicationpublication 2005/0037372, incorporated by reference herein ATB₀Hepatocellular carcinoma 2006/0280725 SEQ ID NOS: 1, 3 of the U.S.patent Liver cancer 2007/0042420 application publication 2007/0042420,incorporated by reference herein CSA-1 Chondrosarcoma 2001/0016649 SEQID NOS: 1-15 of the U.S. patent Pancreatic cancer 2001/0016651application publication 2001/0016651, incorporated by reference hereinSEQ ID NOS: 1-15 of the U.S. patent Pancreatic cancer 2003/0212264application publication 2003/0212264, incorporated by reference hereinSYG972 Breast cancer 2002/0055107 Urb-ctf Breast cancer 2003/0143546BCU399 Breast cancer 2003/0180728 TBX2 Breast cancer 2004/0029185 Cyr61Breast cancer 2004/0086504 DIAPH3 Breast cancer 2005/0054826 SEQ ID NOS:1-24 of the U.S. patent Breast cancer 2007/0134669 applicationpublication 2007/0134669, incorporated by reference herein Humanaspartyl (asparaginyl) beta- CNS cancer 2002/0102263 hydroxylase BEHABCNS cancer 2003/0068661 IL-8 Kaposi's Sarcoma 2003/0096781 SEQ ID NOS:1-278 of the U.S. Hematological cancers 2002/0198362 patent applicationpublication 2002/0198362, incorporated by reference herein BLSA B-cellcancer 2003/0147887 BP1 Leukemia 2003/0171273 DAP-kinase, HOXA9Non-small cell lung cancer 2003/0224509 ARP Clear cell renal carcinoma,2004/0010119 inflammatory disorders Nbk Renal cancer 2005/0053931 CD43Ovarian cancer 2006/0216231 SEQ ID NOS: 1-84 of the U.S. patent Ovariancancer 2007/0054268 application publication 2007/0054268, incorporatedby reference herein β7-hcG, β6-hCG, β6e-hCG, Uterine tumors 2006/0292567β5-hCG, β8-hcG, β3-hCG MTA1s Hormone insensitive 2006/0204957 cancerOld-35, Old-64 Tumor proliferation 2003/0099660 LAGE-1 Cancer 6,794,131CIF150/hTAF_(II)150 Cancer 6,174,679 P65 oncofetal protein Cancer5,773,215 Telomerase Cancer 2002/0025518 CYP1B1 Cancer 2002/005201314-3-3σ Cancer 2002/0102245 NES1 Cancer 2002/0106367 CAR-1 Cancer2002/0119541 HMGI, MAG Cancer 2002/0120120 ELL2 Cancer 2002/0132329Ephrin B2 Cancer 2002/0136726 WAF1 Cancer 2002/0142442 CIF130 Cancer2002/0143154 C35 Cancer 2002/0155447 BMP2 Cancer 2002/0159986 BUB3Cancer 2002/0160403 Polymerase kappa Cancer 2003/0017573 EAG1, EAG2Cancer 2003/0040476 SEQ ID NOS: 18, 20, 22 of the U.S. Cancer2003/0044813 patent application publication 2003/0044813, incorporatedby reference herein HMG I Cancer 2003/0051260 HLTF Cancer 2003/0082526Barx2 Cancer 2003/0087243 SEQ ID NOS: 18, 20, 22, 32, 34, 36 Cancer2003/0108920 of the U.S. patent application publication 2003/0108920,incorporated by reference herein Cables Cancer 2003/0109443 Pp 32r1Cancer 2003/0129631 BMP4 Cancer 2003/0134790 TS10q23.3 Cancer2003/0139324 Nuclear spindle-associating protein Cancer 2003/0157072PFTAIRE Cancer 2003/0166217 SEMA3B Cancer 2003/0166557 MOGp Cancer,multiple sclerosis, 2003/0166898 inflammatory disease Fortilin Cancer2003/0172388 SEQ ID NO: 1 of the U.S. patent Cancer 2003/0215833application publication 2003/0215833, incorporated by reference hereinIGFBP-3 Cancer 2004/0005294 Polyhomeotic 2 Cancer 2004/0006210 PNQALRECancer 2004/0077009 SEQ ID NOS: 1, 3 of the U.S. patent Cancer2004/0086916 application publication 2004/0086916, incorporated byreference herein SCN5A Cancer 2004/0146877 miR15, miR16 Cancer2004/0152112 Headpin Cancer 2004/0180371 PAOh1/SMO Cancer 2004/0229241Hippo, Mst2 Cancer 2005/0053592 PSMA-like Cancer, neurological2005/0064504 disorders JAB1 Cancer 2005/0069918 NF-AT Cancer2005/0079496 P28ING5 Cancer 2005/0097626 MTG16 Cancer 2005/0107313ErbB-2 Cancer 2005/0123538 HDAC9 Cancer 2005/0130146 GPBP Cancer2005/0130227 MG20 Cancer 2005/0153352 KLF6 Cancer 2005/0181374 ARTS1Cancer 2005/0266443 Dock 3 Cancer 2006/0041111 Annexin 8 Cancer2006/0052320 MH15 Cancer 2006/0068411 DELTA-N p73 Cancer 2006/0088825RapR6 Cancer 2006/099676 StarD10 Cancer 2006/0148032 Ciz1 Cancer2006/0155113 HLJ1 Cancer 2006/0194235 RapR7 Cancer 2006/0240021 A34Cancer 2006/0292154 Sef Cancer 2006/0293240 Killin Cancer 2007/0072218SGA-1M Cancer 2007/0128593 TGFβ Type II receptor Cancer 2002/0064786GCA-associated genes Giant cell arteritis 6,743,903 PRV-1 Polycythemiavera 6,686,153 SEQ ID NOS: 2, 4 of the U.S. Pat. No. Ischemia 5,948,6375,948,637, incorporated by reference herein Vezf1 Vascular disorders2002/0023277 MLP Dilatative cardiomyopathy 2002/0042057 VEGIPathological angiogenesis 2002/0111325 PRO256 Cardiovascular disorders2002/0123091 AOP2 Atherosclerosis 2002/0142417 Remodelin Arterialrestenosis, fibrosis 2002/0161211 Phosphodiesterase 4D Stroke2003/0054531 Prostaglandin receptor subtype EP3 Peripheral arterial2003/0157599 occlusive disease CARP Heart disorders 2004/0014706 HOPCongenital heart disease 2004/0029158 SEQ ID NOS: 1-4 of the U.S. patentApoplexy 2004/0087784 application publication 2004/0087784, incorporatedby reference herein PLTP Atherosclerosis, vascular 2006/0252787 disease,hypercholesterolemia, Tangier's disease, familial HDL deficiency diseaseSEQ ID NOS: 1, 3-8, 15, 16 of the Thrombosis 2007/0160996 U.S. patentapplication publication 2007/0160996, incorporated by reference hereinUCP-2 Stroke 2002/0172958 FLJ11011 Fanconi's Anemia 2006/0070134Codanin-1 Anemia 2006/0154331 SEQ ID NOS: 1, 6, 8 of the U.S.Insulin-dependent diabetes 5,763,591 Pat. No. 5,763,591, incorporated bymellitus reference herein Resistin Type II diabetes 2002/0161210Archipelin Diabetes 2003/0202976 SEQ ID NOS: 2, 7, 16, 27 of the U.S.Diabetes, hyperlipidemia 2004/0053397 patent application publication2004/0053397, incorporated by reference herein Neuronatin Metabolicdisorders 2004/0259777 Ncb5or Diabetes 2005/0031605 7B2 Endocrinedisorders 2005/0086709 PTHrP, PEX Metabolic bone diseases 2005/0113303KChIPl Type II diabetes 2005/0196784 SLIT-3 Type II diabetes2006/0141462 CX3CR1 Type II diabetes 2006/0160076 SMAP-2 Diabetes2006/0210974 SEQ ID NOS: 2, 8, 12, 16, 22, 26, Type II diabetes2006/0228706 28, 32 of the U.S. patent application publication2006/0228706, incorporated by reference herein IC-RFX Diabetes2006/0264611 E2IG4 Diabetes, insulin 2007/0036787 resistance, obesitySEQ ID NOS: 2, 8, 10, 14, 18, 24, Diabetes 2007/0122802 26, 30, 34, 38,44, 50, 54, 60, 62, 68, 74, 80, 86, 92, 98, 104, 110 of the U.S. patentapplication publication 2007/0122802, incorporated by reference hereinUCP2 Body weight disorders 2002/0127600 Ob receptor Body weightdisorders 2002/0182676 Ob Body weight disorders 2004/0214214 Dp1Neurodegenerative 2001/0021771 disorders NRG-1 Schizophrenia2002/0045577 Synapsin III Schizophrenia 2002/0064811 NRG1AG1Schizophrenia 2002/0094954 AL-2 Neuronal disorders 2002/0142444 Prolinedehydrogenase Bipolar disorder, major 2002/0193581 depressive disorder,schizophrenia, obsessive compulsive disorder MNR2 Chronicneurodegenerative 2002/0197678 disease ATM Ataxia-telangiectasia2004/0029198 Ho-1 Dementing diseases 2004/0033563 CON202 Schizophrenia2004/0091928 Ataxin-1 Neurodegenerative 2004/0177388 disorders NR3BMotor neuron disorders 2005/0153287 NIPA-1 Hereditary spastic2005/0164228 paraplegia DEPP, adrenomedullin, csdA Schizophrenia2005/0227233 Inf-20 Neurodegenerative 2006/0079675 diseases EOPA Braindevelopment and 2007/0031830 degeneration disorders SERT Autism2007/0037194 FRP-1 Glaucoma 2002/0049177 Serum amyloid A Glaucoma2005/0153927 BMP2 Osteoporosis 2002/0072066 BMPR1A Juvenile polyposis2003/0072758 ACLP Gastroschisis 2003/0084464 Resistin-like molecule βFamilial adenomatous 2003/0138826 polyposis, diabetes, insulinresistance, colon cancer, inflammatory bowel disorder Dlg5 Inflammatorybowel 2006/0100132 disease SEQ ID NOS: 1-82 of the U.S. patentOsteoarthritis 2002/0119452 application publication 2002/0119452,incorporated by reference herein TRANCE Immune system disorders2003/0185820 Matrilin-3 Osteoarthritis 2003/0203380 SynoviolinRheumatoid arthritis 2004/0152871 SEQ ID NOS: 9, 35 of the U.S.Osteoarthritis 2007/0028314 patent application publication 2007/0028314,incorporated by reference herein HIV LTR HIV infection 5,627,023 SHIVAHIV infection 2004/0197770 EBI 1, EBI 2, EBI 3 Epstein Barr virusinfection 2002/0040133 NM23 family Skin/intestinal disorders2002/0034741 SEQ ID NO: 1 of the U.S. patent Psoriasis 2002/0169127application publication 2002/0169127, incorporated by reference hereinEps8 Skin disorders, wound 2003/0180302 healing Beta-10 Thyroid glandpathology 2002/0015981 SEQ ID NO: 2 of the U.S. patent Thyroidconditions 2003/0207403 application publication 2003/0207403,incorporated by reference herein SEQ ID NO: 3 of the U.S. patent Thyroiddisorders 2007/0020275 application publication 2007/0020275,incorporated by reference herein Hair follicle growth factor Alopecia2003/0036174 Corneodesmosin Alopecia 2003/0211065 GCR9 Asthma, lymphoma,2003/0166150 leukemia SEQ ID NO: 1-71 of the U.S. patent Asthma2004/0002084 application publication 2004/0002084, incorporated byreference herein Bg Chediak-Higashi syndrome 2002/0115144 SEQ ID NOS:1-16 of the U.S. patent Endometriosis 2002/0127555 applicationpublication 2002/0127555, incorporated by reference herein FGF23Hypophosphatemic 2005/0156014 disorders BBSR Bardet-Biedl syndrome2003/0152963 MIC-1 Fetal abnormalities, cancer, 2004/0053325inflammatory disorders, miscarriage, premature birth MIA-2 Liver damage2004/0076965 IL-17B Cartilage degenerative 2004/0171109 disordersFormylglycine generating enzyme Multiple sulfatase 2004/0229250deficiency LPLA2 Pulmonary alveolar 2006/0008455 proteinosis CXCL1ORespiratory illnesses 2006/0040329 SEQ ID NOS: 1, 2 of the U.S. patentNephropathy 2006/0140945 application publication 2006/0140945,incorporated by reference herein HFE2A Iron metabolism disease2007/0166711

Once a gene with an expression pattern that is modulated during adisease or disorder is identified, the promoter of the gene may be usedin the gene switch of the invention. The sequence of many genes,including the promoter region, is known in the art and available inpublic databases, e.g., GenBank. Thus, once an appropriate gene isidentified, the promoter sequence can be readily identified andobtained. Another aspect of the present invention is directed towardsidentifying suitable genes whose promoter can be isolated and placedinto a gene switch. The identity of the gene, therefore, may not becritical to specific embodiments of the present invention, provided thepromoter can be isolated and used in subsequent settings orenvironments. The current invention thus includes the use of promotersfrom genes that are yet to be identified. Once suitable genes areidentified, it can be a matter of routine skill or experimentation todetermine the genetic sequences needed for promoter function. Indeed,several commercial protocols exist to aid in the determination of thepromoter region of genes of interest. By way of example, Ding et al.recently elucidated the promoter sequence of the novel Sprouty4 gene(Am. J. Physiol. Lung Cell. Mol. Physiol. 287: L52 (2004), which isincorporated by reference) by progressively deleting the 5′-flankingsequence of the human Sprouty4 gene. Briefly, once the transcriptioninitiation site was determined, PCR fragments were generated usingcommon PCR primers to clone segments of the 5′-flanking segment in aunidirectional manner. The generated segments were cloned into aluciferase reporter vector and luciferase activity was measured todetermine the promoter region of the human Sprouty4 gene.

Another example of a protocol for acquiring and validating genepromoters includes the following steps: (1) acquire diseased andnon-diseased cell/tissue samples of similar/same tissue type; (2)isolate total RNA or mRNA from the samples; (3) perform differentialmicroarray analysis of diseased and non-diseased RNA; (4) identifycandidate disease-specific transcripts; (5) identify genomic sequencesassociated with the disease-specific transcripts; (6) acquire orsynthesize DNA sequence upstream and downstream of the predictedtranscription start site of the disease-specific transcript; (7) designand produce promoter reporter vectors using different lengths of DNAfrom step 6; and (8) test promoter reporter vectors in diseased andnon-diseased cells/tissues, as well as in unrelated cells/tissues.

The source of the promoter that is inserted into the gene switch can benatural or synthetic, and the source of the promoter should not limitthe scope of the invention described herein. In other words, thepromoter may be directly cloned from cells, or the promoter may havebeen previously cloned from a different source, or the promoter may havebeen synthesized.

The gene switch may be any gene switch that regulates gene expression byaddition or removal of a specific ligand. In one embodiment, the geneswitch is one in which the level of gene expression is dependent on thelevel of ligand that is present. Examples of ligand-dependenttranscription factors that may be used in the gene switches of theinvention include, without limitation, members of the nuclear receptorsuperfamily activated by their respective ligands (e.g., glucocorticoid,estrogen, progestin, retinoid, ecdysone, and analogs and mimeticsthereof) and rTTA activated by tetracycline. In one aspect of theinvention, the gene switch is an EcR-based gene switch. Examples of suchsystems include, without limitation, the systems described in U.S. Pat.Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos.2006/0014711, 2007/0161086, and International Published Application No.WO 01/70816. Examples of chimeric ecdysone receptor systems aredescribed in U.S. Pat. No. 7,091,038, U.S. Published Patent ApplicationNos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and2006/0100416, and International Published Application Nos. WO 01/70816,WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, andWO 2005/108617, each of which is incorporated by reference in itsentirety. An example of a non-steroidal ecdysone agonist-regulatedsystem is the RheoSwitch® Mammalian Inducible Expression System (NewEngland Biolabs, Ipswich, Mass.).

In one embodiment, the gene switch comprises a single transcriptionfactor sequence encoding a ligand-dependent transcription factor underthe control of a diagnostic switch promoter. The transcription factorsequence may encode a ligand-dependent transcription factor that is anaturally occurring or an artificial transcription factor. An artificialtranscription factor is one in which the natural sequence of thetranscription factor has been altered, e.g., by mutation of the sequenceor by the combining of domains from different transcription factors. Inone embodiment, the transcription factor comprises a Group H nuclearreceptor LBD. In one embodiment, the Group H nuclear receptor LBD isfrom an EcR, a ubiquitous receptor, an orphan receptor 1, a NER-1, asteroid hormone nuclear receptor 1, a retinoid X receptor interactingprotein-15, a liver X receptor β, a steroid hormone receptor likeprotein, a liver X receptor, a liver X receptor α, a farnesoid Xreceptor, a receptor interacting protein 14, or a farnesol receptor. Inanother embodiment, the Group H nuclear receptor LBD is from an ecdysonereceptor.

The EcR and the other Group H nuclear receptors are members of thenuclear receptor superfamily wherein all members are generallycharacterized by the presence of an amino-terminal transactivationdomain (TD), a DNA binding domain (DBD), and a LBD separated from theDBD by a hinge region. As used herein, the term “DNA binding domain”comprises a minimal polypeptide sequence of a DNA binding protein, up tothe entire length of a DNA binding protein, so long as the DNA bindingdomain functions to associate with a particular response element.Members of the nuclear receptor superfamily are also characterized bythe presence of four or five domains: A/B, C, D, E, and in some membersF (see U.S. Pat. No. 4,981,784 and Evans, Science 240:889 (1988)). The“A/B” domain corresponds to the transactivation domain, “C” correspondsto the DNA binding domain, “D” corresponds to the hinge region, and “E”corresponds to the ligand binding domain. Some members of the family mayalso have another transactivation domain on the carboxy-terminal side ofthe LBD corresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for response elements. These domains may be eithernative, modified, or chimeras of different domains of heterologousreceptor proteins. The EcR, like a subset of the nuclear receptorfamily, also possesses less well-defined regions responsible forheterodimerization properties. Because the domains of nuclear receptorsare modular in nature, the LBD, DBD, and TD may be interchanged.

In another embodiment, the transcription factor comprises a TD, a DBDthat recognizes a response element associated with the reporter genewhose expression is to be modulated; and a Group H nuclear receptor LBD.In certain embodiments, the Group H nuclear receptor LBD comprises asubstitution mutation.

In another embodiment, the gene switch comprises a first transcriptionfactor sequence under the control of a first diagnostic switch promoterand a second transcription factor sequence under the control of a seconddiagnostic switch promoter, wherein the proteins encoded by said firsttranscription factor sequence and said second transcription factorsequence interact to form a protein complex which functions as aligand-dependent transcription factor, i.e., a “dual switch”- or“two-hybrid”-based gene switch. The first and second diagnostic switchpromoters may be the same or different. In this embodiment, the presenceof two different diagnostic switch promoters in the gene switch that arerequired for reporter gene expression enhances the specificity of thediagnostic method (see FIG. 3). FIG. 3 also demonstrates the ability tomodify the diagnostic gene switch to detect any disease or disordersimply by inserting the appropriate diagnostic switch promoters.

In a further embodiment, the first transcription factor sequence isunder the control of a diagnostic switch promoter (e.g., P2 or P3 inFIG. 1) and the second transcription factor sequence is under thecontrol of a constitutive promoter (e.g., P1 in FIG. 1). In thisembodiment, one portion of the ligand-dependent transcription factorwill be constitutively present while the second portion will only besynthesized if the subject has the disease or disorder.

In another embodiment, the first transcription factor sequence is underthe control of a first diagnostic switch promoter (e.g., P1 in FIG. 2)and two or more different second transcription factor sequences areunder the control of different diagnostic switch promoters (e.g., P2 andP3 in FIG. 2). In this embodiment, each of the second transcriptionfactor sequences may have a different DBD that recognizes a differentinducible promoter sequence (e.g., DBD-A binds to inducible promoter Aand DBD-B binds to inducible promoter B). Each of the induciblepromoters may be operably linked to a different reporter gene thatproduces a unique signal. In this manner, multiple diagnoses may be madesimultaneously or a differential diagnosis between two or more possiblediseases may be made.

In one embodiment, the first transcription factor sequence encodes apolypeptide comprising a TD, a DBD that recognizes a response elementassociated with the reporter gene whose expression is to be modulated;and a Group H nuclear receptor LBD, and the second transcription factorsequence encodes a transcription factor comprising a nuclear receptorLBD selected from the group consisting of a vertebrate RXR LBD, aninvertebrate RXR LBD, an ultraspiracle protein LBD, and a chimeric LBDcomprising two polypeptide fragments, wherein the first polypeptidefragment is from a vertebrate RXR LBD, an invertebrate RXR. LBD, or anultraspiracle protein LBD, and the second polypeptide fragment is from adifferent vertebrate RXR LBD, invertebrate RXR LBD, or ultraspiracleprotein LBD.

In another embodiment, the gene switch comprises a first transcriptionfactor sequence encoding a first polypeptide comprising a nuclearreceptor LBD and a DBD that recognizes a response element associatedwith the reporter gene whose expression is to be modulated, and a secondtranscription factor sequence encoding a second polypeptide comprising aTD and a nuclear receptor LBD, wherein one of the nuclear receptor LBDsis a Group H nuclear receptor LBD. In a preferred embodiment, the firstpolypeptide is substantially free of a TD and the second polypeptide issubstantially free of a DBD. For purposes of the invention,“substantially free” means that the protein in question does not containa sufficient sequence of the domain in question to provide activation orbinding activity.

In another aspect of the invention, the first transcription factorsequence encodes a protein comprising a heterodimer partner and a TD andthe second transcription factor sequence encodes a protein comprising aDBD and a LBD.

When only one nuclear receptor LBD is a Group H LBD, the other nuclearreceptor LBD may be from any other nuclear receptor that forms a dimerwith the Group H LBD. For example, when the Group H nuclear receptor LBDis an EcR LBD, the other nuclear receptor LBD “partner” may be from anEcR, a vertebrate RXR, an invertebrate RXR, an ultraspiracle protein(USP), or a chimeric nuclear receptor comprising at least two differentnuclear receptor LBD polypeptide fragments selected from the groupconsisting of a vertebrate RXR, an invertebrate RXR, and a USP (see WO01/70816 A2, International Patent Application No. PCT/US02/05235 and US2004/0096942 A1, incorporated herein by reference in their entirety).The “partner” nuclear receptor ligand binding domain may furthercomprise a truncation mutation, a deletion mutation, a substitutionmutation, or another modification.

In one embodiment, the vertebrate RXR LBD is from a human Homo sapiens,mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pigSus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio,tunicate Polyandrocarpa misakiensis, or jellyfish Tripedalia cysophoraRXR.

In one embodiment, the invertebrate RXR ligand binding domain is from alocust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodidtick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an ixodid tickAmblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celucapugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog(“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphidMyzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-LepidopteranRXR homolog.

In one embodiment, the chimeric RXR LBD comprises at least twopolypeptide fragments selected from the group consisting of a vertebratespecies RXR polypeptide fragment, an invertebrate species RXRpolypeptide fragment, and a non-Dipteranlnon-Lepidopteran invertebratespecies RXR homolog polypeptide fragment. A chimeric RXR ligand bindingdomain for use in the present invention may comprise at least twodifferent species RXR polypeptide fragments, or when the species is thesame, the two or more polypeptide fragments may be from two or moredifferent isoforms of the species RXR polypeptide fragment.

In one embodiment, the chimeric RXR ligand binding domain comprises atleast one vertebrate species RXR polypeptide fragment and oneinvertebrate species RXR polypeptide fragment.

In another embodiment, the chimeric RXR ligand binding domain comprisesat least one vertebrate species RXR polypeptide fragment and onenon-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

The ligand, when combined with the LBD of the nuclear receptor(s), whichin turn are bound to the response element linked to the reporter gene,provides external temporal regulation of expression of the reportergene. The binding mechanism or the order in which the various componentsof this invention bind to each other, that is, for example, ligand toLBD, DBD to response element, TD to promoter, etc., is not critical.

In a specific example, binding of the ligand to the LBD of a Group Hnuclear receptor and its nuclear receptor LBD partner enables expressionof the reporter gene. This mechanism does not exclude the potential forligand binding to the Group H nuclear receptor (GHNR) or its partner,and the resulting formation of active homodimer complexes (e.g.GHNR+GHNR or partner+partner). Preferably, one or more of the receptordomains is varied producing a hybrid gene switch. Typically, one or moreof the three domains, DBD, LBD, and TD, may be chosen from a sourcedifferent than the source of the other domains so that the hybrid genesand the resulting hybrid proteins are optimized in the chosen host cellor organism for transactivating activity, complementary binding of theligand, and recognition of a specific response element. In addition, theresponse element itself can be modified or substituted with responseelements for other DNA binding protein domains such as the GAL-4 proteinfrom yeast (see Sadowski et al., Nature 335:563 (1988)) or LexA proteinfrom Escherichia coli (see Brent et al., Cell 43:729 (1985)), orsynthetic response elements specific for targeted interactions withproteins designed, modified, and selected for such specific interactions(see, for example, Kim et al., Proc. Natl. Acad. Sci. USA, 94:3616(1997)) to accommodate hybrid receptors.

The functional EcR complex may also include additional protein(s) suchas immunophilins. Additional members of the nuclear receptor family ofproteins, known as transcriptional factors (such as DHR38 or betaFTZ-1),may also be ligand dependent or independent partners for EcR, USP,and/or RXR. Additionally, other cofactors may be required such asproteins generally known as coactivators (also termed adapters ormediators). These proteins do not bind sequence-specifically to DNA andare not involved in basal transcription. They may exert their effect ontranscription activation through various mechanisms, includingstimulation of DNA-binding of activators, by affecting chromatinstructure, or by mediating activator-initiation complex interactions.Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as thepromiscuous coactivator C response element B binding protein, CBP/p300(for review see Glass et al., Curr. Opin. Cell Biol. 9:222 (1997)).Also, protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the unliganded EcR to silence theactivity at the response element. Current evidence suggests that thebinding of ligand changes the conformation of the receptor, whichresults in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N-CoR and SMRT (for review, see Horwitzet al., Mol Endocrinol. 10:1167 (1996)). These cofactors may either beendogenous within the cell or organism, or may be added exogenously astransgenes to be expressed in either a regulated or unregulated fashion.

The reporter gene may be any gene that encodes a detectable protein. Theprotein may be secreted or non-secreted. In one embodiment, the proteinis one that can be assayed using various standard assay methods, e.g.,immunoassays (such as those immunofluorescent antibodies), colorimetricassays, fluorescent assays, or luminescent assays. Examples of suitablereporter genes include, without limitation, luciferase, greenfluorescent protein, β-galactosidase, β-glucuronidase, thymidine kinase,and chloramphenicol acetyltransferase.

The reporter gene is operably linked to a promoter comprising at leastone response element that is recognized by the DBD of theligand-dependent transcription factor encoded by the gene switch. In oneembodiment, the promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore copies of the response element. Promoters comprising the desiredresponse elements may be naturally occurring promoters or artificialpromoters created using techniques that are well known in the art, e.g.,one or more response elements operably linked to a minimal promoter.

To introduce the polynucleotides into the cells, a vector can be used.The vector may be, for example, a plasmid vector or a single- ordouble-stranded RNA or DNA viral vector. Such vectors may be introducedinto cells by well-known techniques for introducing DNA and RNA intocells. Viral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells. As used herein, the term “host cell”or “host” is used to mean a cell of the present invention that isharboring one or more polynucleotides of the invention.

Thus, at a minimum, the vectors must include the polynucleotides of theinvention. Other components of the vector may include, but are notlimited to, selectable markers, chromatin modification domains,additional promoters driving expression of other polypeptides that mayalso be present on the vector (e.g., a lethal polypeptide), genomicintegration sites, recombination sites, and molecular insertion pivots.The vectors may comprise any number of these additional elements, eitherwithin or not within the polynucleotides, such that the vector can betailored to the specific goals of the diagnostic methods desired.

In one embodiment of the present invention, the vectors that areintroduced into the cells further comprise a “selectable marker gene”which, when expressed, indicates that the diagnostic gene switchconstruct of the present invention has been integrated into the genomeof the host cell. In this manner, the selector gene can be a positivemarker for the genome integration. While not critical to the methods ofthe present invention, the presence of a selectable marker gene allowsthe practitioner to select for a population of live cells where thevector construct has been integrated into the genome of the cells. Thus,certain embodiments of the present invention comprise selecting cellswhere the vector has successfully been integrated. As used herein, theterm “select” or variations thereof, when used in conjunction withcells, is intended to mean standard, well-known methods for choosingcells with a specific genetic make-up or phenotype. Typical methodsinclude, but are not limited to, culturing cells in the presence ofantibiotics, such as G418, neomycin and ampicillin. Other examples ofselectable marker genes include, but are not limited to, genes thatconfer resistance to dihydrofolate reductase, hygromycin, ormycophenolic acid. Other methods of selection include, but are notlimited to, a selectable marker gene that allows for the use ofthymidine kinase, hypoxanthine-guanine phosphoribosyltransferase oradenine phosphoribosyltransferase as selection agents. Cells comprisinga vector construct comprising an antibiotic resistance gene or geneswould then be capable of tolerating the antibiotic in culture. Likewise,cells not comprising a vector construct comprising an antibioticresistance gene or genes would not be capable of tolerating theantibiotic in culture.

As used herein, a “chromatin modification domain” (CMD) refers tonucleotide sequences that interact with a variety of proteins associatedwith maintaining and/or altering chromatin structure, such as, but notlimited to, DNA insulators. See Ciavatta et al., Proc. Nat'l Acad. Sci.U.S.A., 103:9958 (2006), which is incorporated by reference herein.Examples of CMDs include, but are not limited to, the chicken β-globulininsulator and the chicken hypersensitive site 4 (cHS4). The use ofdifferent CMD sequences between one or more gene programs (i.e., apromoter, coding sequence, and 3′ regulatory region), for example, canfacilitate the use of the differential CMD DNA sequences as “minihomology arms” in combination with various microorganism or in vitrorecombineering technologies to “swap” gene programs between existingmultigenic and monogenic shuttle vectors. Other examples of chromatinmodification domains are known in the art or can be readily identified.

Particular vectors for use with the present invention are expressionvectors that code for proteins or portions thereof. Generally, suchvectors comprise cis-acting control regions effective for expression ina host operatively linked to the polynucleotide to be expressed.Appropriate trans-acting factors are supplied by the host, supplied by acomplementing vector or supplied by the vector itself upon introductioninto the host.

A great variety of expression vectors can be used to express proteins.Such vectors include chromosomal, episomal and virus-derived vectors,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromyeast episomes, from yeast chromosomal elements, from viruses such asadeno-associated viruses, lentiviruses, baculoviruses, papova viruses,such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. All maybe used for expression in accordance with this aspect of the presentinvention. Generally, any vector suitable to maintain, propagate orexpress polynucleotides or proteins in a host may be used for expressionin this regard.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s) including, for instance, apromoter to direct mRNA transcription. Representatives of additionalpromoters include, but are not limited to, constitutive promoters andtissue specific or inducible promoters. Examples of constitutiveeukaryotic promoters include, but are not limited to, the promoter ofthe mouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen. 1:273(1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982));the SV40 early promoter (Benoist et al., Nature 290:304 (1981)); and thevaccinia virus promoter. All of the above listed references areincorporated by reference herein. Additional examples of the promotersthat could be used to drive expression of a protein include, but are notlimited to, tissue-specific promoters and other endogenous promoters forspecific proteins, such as the albumin promoter (hepatocytes), aproinsulin promoter (pancreatic beta cells) and the like. In general,expression constructs will contain sites for transcription, initiationand termination and, in the transcribed region, a ribosome binding sitefor translation. The coding portion of the mature transcripts expressedby the constructs may include a translation initiating AUG at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate,as well as engender expression. Generally, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

Examples of eukaryotic vectors include, but are not limited to, pW-LNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV,pMSG and pSVL available from Amersham Pharmacia Biotech; andpCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, pCMV-EGFP availablefrom Clontech. Many other vectors are well-known and commerciallyavailable.

Particularly useful vectors, which comprise molecular insertion pivotsfor rapid insertion and removal of elements of gene programs, aredescribed in United States Published Patent Application No.2004/0185556, U.S. patent application Ser. No. 11/233,246 andInternational Published Application Nos. WO 2005/040336 and WO2005/116231, all of which are incorporated by reference. An example ofsuch vectors is the UltraVector™ Production System (Intrexon Corp.,Blacksburg, Va.), as described in WO 2007/038276, incorporated herein byreference. As used herein, a “gene program” is a combination of geneticelements comprising a promoter (P), an expression sequence (E) and a 3′regulatory sequence (3), such that “PE3” is a gene program. The elementswithin the gene program can be easily swapped between molecular pivotsthat flank each of the elements of the gene program. A molecular pivot,as used herein, is defined as a polynucleotide comprising at least twonon-variable rare or uncommon restriction sites arranged in a linearfashion. In one embodiment, the molecular pivot comprises at least threenon-variable rare or uncommon restriction sites arranged in a linearfashion. Typically any one molecular pivot would not include a rare oruncommon restriction site of any other molecular pivot within the samegene program. Cognate sequences of greater than 6 nucleotides upon whicha given restriction enzyme acts are referred to as “rare” restrictionsites. There are, however, restriction sites of 6 bp that occur moreinfrequently than would be statistically predicted, and these sites andthe endonucleases that cleave them are referred to as “uncommon”restriction sites. Examples of either rare or uncommon restrictionenzymes include, but are not limited to, AsiS I, Pac I, Sbf I, Fse I,Asc I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, SgrAI, AflIII, Pvu I, Ngo MIV, Ase I, Flp I, Pme I, Sda I, Sgf I, Srf I,and Sse8781 I.

The vector may also comprise restriction sites for a second class ofrestriction enzymes called homing endonuclease (HE) enzymes. HE enzymeshave large, asymmetric restriction sites (12-40 base pairs), and theirrestriction sites are infrequent in nature. For example, the HE known asI-SceI has an 18 bp restriction site (5′TAGGGATAACAGGGTAAT3′ (SEQ IDNO:4)), predicted to occur only once in every 7×10¹⁰ base pairs ofrandom sequence. This rate of occurrence is equivalent to only one sitein a genome that is 20 times the size of a mammalian genome. The rarenature of HE sites greatly increases the likelihood that a geneticengineer can cut a gene program without disrupting the integrity of thegene program if HE sites were included in appropriate locations in acloning vector plasmid.

Selection of appropriate vectors and promoters for expression in a hostcell is a well-known procedure, and the requisite techniques for vectorconstruction and introduction into the host, as well as its expressionin the host are routine skills in the art.

The introduction of the polynucleotides into the cells can be atransient transfection, stable transfection, or can be a locus-specificinsertion of the vector. Transient and stable transfection of thevectors into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology(1986); Keown et al., 1990, Methods Enzymol. 185: 527-37; Sambrook etal., 2001, Molecular Cloning, A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory Press, N.Y., which are hereby incorporated byreference. These stable transfection methods result in random insertionof the vector into the genome of the cell. Further, the copy number andorientation of the vectors are also, generally speaking, random.

In one embodiment of the invention, the vector is inserted into abio-neutral site in the genome. A bio-neutral site is a site in thegenome where insertion of the polynucleotides interferes very little, ifany, with the normal function of the cell. Bio-neutral sites may beanalyzed using available bioinformatics. Many bio-neutral sites areknown in the art, e.g., the ROSA-equivalent locus. Other bio-neutralsites may be identified using routine techniques well known in the art.Characterization of the genomic insertion site(s) is performed usingmethods known in the art. To control the location, copy number and/ororientation of the polynucleotides when introducing the vector into thecells, methods of locus-specific insertion may be used. Methods oflocus-specific insertion are well-known in the art and include, but arenot limited to, homologous recombination and recombinase-mediated genomeinsertion. Of course, if locus-specific insertion methods are to be usedin the methods of the present invention, the vectors may compriseelements that aid in this locus-specific insertion, such as, but notlimited to, homologous recombination. For example, the vectors maycomprise one, two, three, four or more genomic integration sites (GISs).As used herein, a “genomic integration site” is defined as a portion ofthe vector sequence which nucleotide sequence is identical or nearlyidentical to portions of the genome within the cells that allows forinsertion of the vector in the genome. In particular, the vector maycomprise two genomic insertion sites that flank at least thepolynucleotides. Of course, the GISs may flank additional elements, oreven all elements present on the vector.

In another embodiment, locus-specific insertion may be carried out byrecombinase-site specific gene insertion. Briefly, bacterial recombinaseenzymes, such as, but not limited to, PhiC31 integrase can act on“pseudo” recombination sites within the human genome. These pseudorecombination sites can be targets for locus-specific insertion usingthe recombinases. Recombinase-site specific gene insertion is describedin Thyagarajan et al., Mol. Cell Biol. 21:3926 (2001), which is herebyincorporated by reference. Other examples of recombinases and theirrespective sites that may be used for recombinase-site specific geneinsertion include, but are not limited to, serine recombinases such asR4 and TP901-1 and recombinases described in WO 2006/083253,incorporated herein by reference.

In a further embodiment, the vector may comprise a chemo-resistancegene, e.g., the multidrug resistance gene mdr1, dihydrofolate reductase,or O⁶-alkylguanine-DNA alkyltransferase. The chemo-resistance gene maybe under the control of a constitutive (e.g., CMV) or inducible (e.g.,RheoSwitch®) promoter. In this embodiment, if it is desired to treat adisease diagnosed in a subject while maintaining the modified cellswithin the subject, a clinician may apply a chemotherapeutic agent todestroy diseased cells while the modified cells would be protected fromthe agent due to expression of a suitable chemo-resistance gene and maycontinue to be used for monitoring of the progression of the disease oreffectiveness of the treatment. By placing the chemo-resistance geneunder an inducible promoter, the unnecessary expression of thechemo-resistance gene can be avoided, yet it will still be available incase continued diagnosis is desired during treatment. If the modifiedcells themselves become diseased, they could still be destroyed byinducing expression of a lethal polypeptide as described below.

The methods of the invention are carried out by introducing thepolynucleotides encoding the gene switch and the reporter gene intocells of a subject. Any method known for introducing a polynucleotideinto a cell known in the art, such as those described above, can beused.

In an alternative embodiment, the polynucleotides encoding the geneswitch and the reporter gene are introduced into non-autologous cells,e.g., cells that are allogeneic or xenogeneic to the subject.

When the polynucleotides are to be introduced into cells ex vivo, thecells may be obtained from a subject by any technique known in the art,including, but not limited to, biopsies, scrapings, and surgical tissueremoval. The isolated cells may be cultured for a sufficient amount oftime to allow the polynucleotides to be introduced into the cells, e.g.,2, 4, 6, 8, 10, 12, 18, 24, 36, 48, hours or more. Methods for culturingprimary cells for short periods of time are well known in the art. Forexample, cells may be cultured in plates (e.g., in microwell plates)either attached or in suspension.

For ex vivo diagnosis methods, cells are isolated from a subject andcultured under conditions suitable for introducing the polynucleotidesinto the cells. Once the polynucleotides have been introduced into thecells, the cells are incubated for a sufficient period of time to allowthe ligand-dependent transcription factor to be expressed, e.g., 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours or more. If expressionof the ligand-dependent transcription factor is increased or decreasedcompared to control levels (i.e., if the subject has the disease ordisorder and the promoters controlling expression of the transcriptionfactor are activated or deactivated), the presence and/or level of theligand-dependent transcription factor is detected by the addition ofligand, leading to expression of the reporter gene at a levelcorresponding to the level of the ligand-dependent transcription factor.The ligand may be added to the cells at any time before, during or afterintroduction of the polynucleotides into the cells. The optimal timingof ligand administration can be determined for each type of cell anddisease or disorder using only routine techniques. In one embodiment,the ligand may be added, reporter gene expression determined, the ligandremoved, and the process repeated one or more times to obtain multiplediagnostic measurements of the cells. In another embodiment, ligand iscontinuously present and reporter gene expression is measuredperiodically.

The first in vivo diagnostic embodiment of the invention (modificationof isolated cells followed by reintroduction of the cells to thesubject) may be used where the ex vivo method using isolated cells isinsufficient, e.g., where circulating factors are necessary fordiagnostic switch promoter activity to occur. In this embodiment, cellsare isolated from a subject and the polynucleotides are introduced intothe cells in culture as described above. At some point after theintroduction of the polynucleotides into the cells, the cells areintroduced back into the subject. Reintroduction may be carried out byany method known in the art, e.g., intravenous infusion or directinjection into a tissue or cavity. In one embodiment, the presence ofthe polynucleotides in the cells is determined prior to introducing thecells back into the subject. In another embodiment, cells containing thepolynucleotides are selected (e.g., based on the presence of aselectable marker in the polynucleotides) and only those cellscontaining the polynucleotides are reintroduced into the subject. Afterthe cells are reintroduced to the subject, ligand is administered to thesubject and reporter gene expression is assayed. The ligand may beadministered by any suitable method, either systemically (e.g., orally,intravenously) or locally (e.g., intraperitoneally, intrathecally,intraventricularly, direct injection into the tissue or organ where thecells were reintroduced). The optimal timing of ligand administrationcan be determined for each type of cell and disease or disorder usingonly routine techniques. In one embodiment, the ligand may beadministered, reporter gene expression determined, the ligand removed,and the process repeated one or more times to obtain multiple diagnosticmeasurements of the cells. In another embodiment, ligand is continuouslyadministered and reporter gene expression is measured periodically. Thedetection of reporter gene expression after ligand is administered canoccur in vivo or ex vivo. For example, if the reporter gene encodes asecreted protein that circulates in the blood, detection of the proteincan occur in a blood sample removed from the patient. If the reportergene encodes a protein that produces a luminescent or fluorescentsignal, the signal may be detected in vivo. In another embodiment, asample of the modified cells can be removed and expression of thereporter gene detected ex vivo.

The second in vivo diagnostic embodiment of the invention involvesdirect in vivo introduction of the polynucleotides into the cells of thesubject. The polynucleotides may be introduced into the subjectsystemically or locally (e.g., at the site of the suspected disease ordisorder). Once the polynucleotides have been introduced to the subject,the ligand may be administered and reporter gene expression assayed. Theligand may be administered by any suitable method, either systemically(e.g., orally, intravenously) or locally (e.g., intraperitoneally,intrathecally, intraventricularly, direct injection into the tissue ororgan where the suspected disease or disorder is occurring). The optimaltiming of ligand administration can be determined for each type of celland disease or disorder using only routine techniques. In oneembodiment, the ligand may be added, reporter gene expressiondetermined, the ligand removed, and the process repeated one or moretimes to obtain multiple diagnostic measurements of the cells containingthe polynucleotides. In another embodiment, ligand is continuouslyadministered and reporter gene expression is measured periodically. Thedetection of reporter gene expression after ligand is administered canoccur in vivo or ex vivo. For example, if the reporter gene encodes asecreted protein that circulates in the blood, detection of the proteincan occur in a blood sample removed from the patient. If the reportergene encodes a protein that produces a luminescent or fluorescentsignal, the signal may be detected in vivo. In another embodiment, asample of the modified cells can be removed and expression of thereporter gene detected ex vivo.

When non-autologous cells are used in the diagnostic methods, the cellsmay be obtained from any source, e.g., other subjects, cell lines, oranimals. The non-autologous cells may be any cells that are viable aftertransplantation, such as fibroblasts or stem cells (e.g., embryonic stemcells, hematopoietic stem cells). The non-autologous cells are isolatedand the polynucleotides are introduced into the cells in culture asdescribed above. At some point after the introduction of thepolynucleotides into the cells, the cells are introduced into thesubject. Introduction may be carried out by any method known in the art,e.g., intravenous infusion or direct injection into a tissue or cavity.In one embodiment, the presence of the polynucleotides in the cells isdetermined prior to introducing the cells back into the subject. Inanother embodiment, cells containing the polynucleotides are selected(e.g., based on the presence of a selectable marker in thepolynucleotides) and only those cells containing the polynucleotides areintroduced into the subject. In one embodiment, the non-autologous cellsare surrounded by a barrier (e.g., encapsulated) prior to introductioninto the subject. After the cells are introduced to the subject, ligandis administered to the subject and reporter gene expression is assayed.The ligand may be administered by any suitable method, eithersystemically (e.g., orally, intravenously) or locally (e.g.,intraperitoneally, intrathecally, intraventricularly, direct injectioninto the tissue or organ where the cells were introduced). The optimaltiming of ligand administration can be determined for each type of celland disease or disorder using only routine techniques. In oneembodiment, the ligand may be administered, reporter gene expressiondetermined, the ligand removed, and the process repeated one or moretimes to obtain multiple diagnostic measurements of the cells. Inanother embodiment, ligand is continuously administered and reportergene expression is measured periodically. The detection of reporter geneexpression after ligand is administered can occur in vivo or ex vivo.For example, if the reporter gene encodes a secreted protein thatcirculates in the blood, detection of the protein can occur in a bloodsample removed from the patient. If the reporter gene encodes a proteinthat produces a luminescent or fluorescent signal, the signal may bedetected in vivo. In another embodiment, a sample of the modified cellscan be removed and expression of the reporter gene detected ex vivo.

In all in vivo embodiments, the polynucleotides or vector comprising thepolynucleotides may comprise a sequence encoding a lethal polypeptidethat can be turned on to express a product that will kill a cellcontaining the polynucleotides or vector. Lethal polypeptide expressioncan be used to eliminate the modified cells from a subject, eitherbecause diagnostic tests are no longer needed or because of a problemwith the modified cells (e.g., hyperproliferation or toxicity). A lethalpolypeptide, as used herein, is a polypeptide that, when expressed, islethal to the cell that expresses the polypeptide, either because thepolypeptide itself is lethal or the polypeptide produces a compound thatis lethal. As used herein, a lethal polypeptide includes polypeptidesthat induce cell death in any fashion, including but not limited to,necrosis, apoptosis and cytotoxicity. Examples of lethal polypeptidesinclude, but are not limited to, apoptosis inducing tumor suppressorgenes such as, but not limited to, p53, Rb and BRCA-1, toxins such asdiphtheria toxin (DTA), shigella neurotoxin, botulism toxin, tetanustoxin, cholera toxin, CSE-V2 and other variants of scorpion proteintoxins to name a few, suicide genes such as cytosine deaminase andthymidine kinase, and cytotoxic genes, e.g., tumor necrosis factor,interferon-alpha. The present invention is not limited by the identityof the lethal polypeptide, provided that the polypeptide is capable ofbeing lethal to the cell in which it is expressed. If the modified cellsare short-lived cells (e.g., cells with a limited lifespan (e.g., about10 days or less, such as dendritic cells), it may not be necessary toinclude a lethal polypeptide in the polynucleotides or vector as thecells will be naturally removed over a short period of time.

Another aspect of the invention relates to methods of monitoringprogression of a disease or disorder by administering to cells of thesubject the diagnostic gene switches of the invention and measuringreporter gene expression to monitor progression of the disease ordisorder. In one embodiment, the invention relates to methods ofmonitoring the progression of a disease or disorder in a subject,comprising:

-   (a) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said disease or disorder in said subject.

Progression may be indicated by increasing or decreasing reporter geneexpression depending on whether the diagnostic switch promoters areresponsive to factor(s) that increase or decrease during progression ofthe disease or disorder. These methods may be carried out using any ofthe variants of the diagnostic methods described above (i.e., ex vivocells, modification of cells ex vivo followed by reintroduction of thecells in vivo, or in vivo). A disease or disorder is monitored bymeasuring reporter gene expression at least twice as an indication ofthe state of the disease or disorder and noting any change in the levelof expression. In one embodiment, the monitoring can be carried out byexposing the cells to ligand continuously and measuring reporter geneexpression intermittently. In another embodiment, the monitoring can becarried out by exposing cells to ligand intermittently and measuringreporter gene expression during each exposure.

One embodiment of the invention relates to methods of preparing modifiedcells for monitoring the progression of a disease or disorder in asubject, comprising introducing into cells of said subject (1) apolynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, operably linked to a diagnostic switch promoter, wherein theactivity of the promoter is modulated during said disease or disorder,and (2) a polynucleotide encoding a reporter gene linked to a promoterwhich is activated by said ligand-dependent transcription factor, toproduce modified cells.

Another embodiment of the invention relates to methods of monitoring theprogression of a disease or disorder in a subject, comprising:

-   (a) administering ligand to modified cells of said subject; and-   (b) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said disease or disorder in said subject; and

wherein said modified cells of said subject comprise (1) apolynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, operably linked to a diagnostic switch promoter, wherein theactivity of the promoter is modulated during said disease or disorder,and (2) a polynucleotide encoding a reporter gene linked to a promoterwhich is activated by said ligand-dependent transcription factor.

In a further embodiment, the methods of monitoring progression of adisease or disorder are carried out using non-autologous cells, e.g.,cells that are allogeneic or xenogeneic to the subject, and the modifiednon-autologous cells are introduced into the subject. In one embodiment,the non-autologous cells are surrounded by a barrier (e.g.,encapsulated) prior to being introduced into the subject. One embodimentof the invention relates to methods of monitoring the progression of adisease or disorder in a subject, comprising:

-   (a) introducing into non-autologous cells (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) introducing said modified cells into said subject;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said disease or disorder in said subject.

A further aspect of the invention relates to methods of monitoring theeffectiveness of a treatment for a disease or disorder in a subject,comprising administering to the subject a treatment and carrying out thediagnostic methods of the invention at least twice to determine ifreporter gene expression is increasing, decreasing, or remaining thesame. In one embodiment, the invention relates to methods of monitoringthe effectiveness of a treatment for a disease or disorder in a subject,comprising:

-   (a) administering said treatment to said subject;-   (b) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the effectiveness of said treatment.

A change in the level of expression of the reporter gene after thetreatment is administered is an indication of the effectiveness of thetreatment. A decrease in reporter gene expression indicates thetreatment is effective if the diagnostic switch promoter(s) areresponsive to factor(s) that are elevated in the disease or disorder. Anincrease in reporter gene expression indicates the treatment iseffective if the diagnostic switch promoter(s) are responsive tofactor(s) that are reduced in the disease or disorder. If reporter geneexpression does not change after administration of the treatment, it mayindicate that the treatment has halted the progression of the disease ordisorder. These methods may be carried out using any of the variants ofthe diagnostic methods described above (i.e., ex vivo cells,modification of cells ex vivo followed by reintroduction of the cells invivo, in vivo).

For the ex vivo embodiment of the invention, cells may be isolated fromthe subject before treatment to determine baseline levels of reportergene expression. After the treatment is administered to the subject,cells may be isolated from the subject at various intervals to determinereporter gene expression.

For the in vivo embodiments of the invention, modified cells or thepolynucleotides can be introduced into a subject before, during, orafter administration of the treatment. If the cells or polynucleotidesare administered prior to the treatment, a baseline level of reportergene expression can be obtained.

The measurement of reporter gene expression may be carried out ex vivoor in vivo. In one embodiment, the monitoring can be carried out byexposing the cells to ligand continuously and measuring reporter geneexpression intermittently. In another embodiment, the monitoring can becarried out by exposing cells to ligand intermittently and measuringreporter gene expression during each exposure.

In one embodiment, one or both of the polynucleotides encoding a geneswitch and a reporter gene may be part of a therapeutic vector that isbeing administered to a subject (e.g., a vector encoding a therapeuticprotein or nucleic acid for gene therapy). In this embodiment, thetherapeutic treatment and the diagnostic test for monitoringeffectiveness of the treatment are administered together in one unit,ensuring that all cells that receive the treatment also receive thediagnostic gene switch.

One embodiment of the invention relates to methods of preparing modifiedcells for monitoring the effectiveness of a treatment for a disease ordisorder in a subject, comprising introducing into cells of said subject(1) a polynucleotide encoding a gene switch, said gene switch comprisingat least one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, operably linked to a diagnostic switch promoter, wherein theactivity of the promoter is modulated during said disease or disorder,and (2) a polynucleotide encoding a reporter gene linked to a promoterwhich is activated by said ligand-dependent transcription factor, toproduce modified cells.

Another embodiment of the invention relates to methods of monitoring theeffectiveness of a treatment for a disease or disorder in a subject,comprising:

-   (a) administering said treatment to said subject;-   (b) administering ligand to modified cells of said subject; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the effectiveness of said treatment; and

wherein said modified cells of said subject comprise (1) apolynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, operably linked to a diagnostic switch promoter, wherein theactivity of the promoter is modulated during said disease or disorder,and (2) a polynucleotide encoding a reporter gene linked to a promoterwhich is activated by said ligand-dependent transcription factor.

In a further embodiment, the methods of monitoring the effectiveness ofa treatment for a disease or disorder are carried out usingnon-autologous cells, e.g., cells that are allogeneic or xenogeneic tothe subject, and the modified non-autologous cells are introduced intothe subject. In one embodiment, the non-autologous cells are surroundedby a barrier (e.g., encapsulated) prior to being introduced into thesubject. In one embodiment the invention relates to methods ofmonitoring the effectiveness of a treatment for a disease or disorder ina subject, comprising:

-   (a) introducing into non-autologous cells (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    said disease or disorder, and (2) a polynucleotide encoding a    reporter gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) introducing said modified cells into said subject;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the effectiveness of said treatment.

Another aspect of the invention relates to methods of monitoring thepotential toxicity of an administered treatment for a disease ordisorder in a subject. In one embodiment, the invention relates tomethods of monitoring the potential toxicity of an administeredtreatment for a disease or disorder in a subject, comprising:administering said treatment to said subject;

-   (a) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, linked to a diagnostic switch promoter,    wherein the activity of the promoter is modulated by factors found    in cells that are being exposed to toxic conditions, and (2) a    polynucleotide encoding a reporter gene linked to a promoter which    is activated by said ligand-dependent transcription factor, to    produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the toxicity of said treatment.

In one embodiment, this aspect involves polynucleotides in which thetranscription factor sequence(s) are under the control of promoters thatare regulated by factors found in cells that are being exposed to toxicconditions, e.g., cells that are stressed or dying. Examples include,without limitation, promoters responsive to apoptosis signals, necrosissignals, hypoxia, reactive oxygen species, DNA or chromatinmodification, protein degradation, oxidative/reductive state, changes inpH, etc. Suitable stress promoters include those disclosed in U.S.Published Application No. 2003/0027127 (incorporated herein byreference) and include, without limitation, promoters from the followinggenes: CYP1A1, GST Ya, GADD45, GRP78, JUN, FOS, XHF, HSP70, MT IIA,GADD153, ALDH 1, HMO, CRE, XRE, NFκBRE, RARE, ThRE, PPRE, TRE, ERE, andp53RE. Suitable apoptosis-responsive promoters include, withoutlimitation, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DRS, DR6, FADD,RIP, TNFα, Fas ligand, TRAILR1, TRAILR2, TRAILR3, Bcl-2, p53, BAX, BAD,Akt, CAD, PI3 kinase, PP1, and caspase proteins. Detection of anincrease in reporter gene expression following administration of atreatment is an indication that the treatment is harmful to the cells.By making the gene switch responsive to stress or death signals, it canbe used to monitor the effects of a treatment and detect toxic effectson the cellular level long before the subject exhibits overt symptoms oftoxicity.

These methods may be carried out using any of the variants of thediagnostic methods described above (i.e., ex vivo cells, modification ofcells ex vivo followed by reintroduction of the cells in vivo, in vivo).

For the ex vivo embodiment of the invention, cells may be isolated fromthe subject before treatment to determine baseline levels of reportergene expression. After the treatment is administered to the subject,cells may be isolated from the subject at various intervals to determinereporter gene expression.

For the in vivo embodiments of the invention, modified cells or thepolynucleotides can be introduced into a subject before, during, orafter administration of the treatment. If the cells or polynucleotidesare administered prior to the treatment, a baseline level of reportergene expression can be obtained.

The measurement of reporter gene expression may be carried out ex vivoor in vivo. In one embodiment, the monitoring can be carried out byexposing the cells to ligand continuously and measuring reporter geneexpression intermittently. In another embodiment, the monitoring can becarried out by exposing cells to ligand intermittently and measuringreporter gene expression during each exposure.

One embodiment of the invention relates to methods of preparing modifiedcells for monitoring the potential toxicity of an administered treatmentfor a disease or disorder in a subject, comprising introducing intocells of said subject (1) a polynucleotide encoding a gene switch, saidgene switch comprising at least one transcription factor sequence,wherein said at least one transcription factor sequence encodes aligand-dependent transcription factor, linked to a diagnostic switchpromoter, wherein the activity of the promoter is modulated by factorsfound in cells that are being exposed to toxic conditions, and (2) apolynucleotide encoding a reporter gene linked to a promoter which isactivated by said ligand-dependent transcription factor, to producemodified cells.

Another embodiment of the invention relates to methods of monitoring thepotential toxicity of an administered treatment for a disease ordisorder in a subject, comprising:

-   (a) administering said treatment to said subject;-   (b) administering ligand to modified cells of said subject; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the toxicity of said treatment; and

wherein said modified cells of said subject comprise (1) apolynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, linked to a diagnostic switch promoter, wherein the activity ofthe promoter is modulated by factors found in cells that are beingexposed to toxic conditions, and (2) a polynucleotide encoding areporter gene linked to a promoter which is activated by saidligand-dependent transcription factor.

In a further embodiment, the methods of monitoring the potentialtoxicity of an administered treatment for a disease or disorder arecarried out using non-autologous cells, e.g., cells that are allogeneicor xenogeneic to the subject, and the modified non-autologous cells areintroduced into the subject. In one embodiment, the non-autologous cellsare surrounded by a barrier (e.g., encapsulated) prior to beingintroduced into the subject. In one embodiment, the invention relates tomethods of monitoring the potential toxicity of an administeredtreatment for a disease or disorder in a subject, comprising:

-   (a) introducing into non-autologous cells (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated by    factors found in cells that are being exposed to toxic conditions,    and (2) a polynucleotide encoding a reporter gene linked to a    promoter which is activated by said ligand-dependent transcription    factor, to produce modified cells;-   (b) introducing said modified cells into said subject;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates the toxicity of said treatment.

In another embodiment of the invention, the polynucleotide comprisestranscription factor sequence(s) that are under the control of promotersthat are activated by the factor which is being administered as thetreatment (e.g., gene therapy treatment with a therapeutic protein ornucleic acid). By making the gene switch responsive to the administeredtreatment, it can be used to monitor expression of the gene therapytreatment and detect undesirably high or low levels of the treatmentlong before the subject exhibits overt symptoms of overexpression orunderexpression of the therapeutic factor. In one embodiment, theinvention relates to methods of monitoring the level of a factor that isbeing administered to a subject for treatment for a disease or disorder,comprising: administering said treatment to said subject;

-   (a) introducing into cells of said subject (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, linked to a diagnostic switch promoter,    wherein the activity of the promoter is modulated by said factor    that is being administered for treatment, and (2) a polynucleotide    encoding a reporter gene linked to a promoter which is activated by    said ligand-dependent transcription factor, to produce modified    cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression;

wherein the level of expression of said reporter gene indicates thelevel of the factor being administered for treatment.

These methods may be carried out using any of the variants of thediagnostic methods described above (i.e., ex vivo cells, modification ofcells ex vivo followed by reintroduction of the cells in vivo, in vivo).

For the ex vivo embodiment of the invention, cells may be isolated fromthe subject before treatment to determine baseline levels of reportergene expression. After the treatment is administered to the subject,cells may be isolated from the subject at various intervals to determinereporter gene expression.

For the in vivo embodiments of the invention, modified cells or thepolynucleotides can be introduced into a subject before, during, orafter administration of the treatment. If the cells or polynucleotidesare administered prior to the treatment, a baseline level of reportergene expression can be obtained.

The measurement of reporter gene expression may be carried out ex vivoor in vivo. In one embodiment, the monitoring can be carried out byexposing the cells to ligand continuously and measuring reporter geneexpression intermittently. In another embodiment, the monitoring can becarried out by exposing cells to ligand intermittently and measuringreporter gene expression during each exposure.

One embodiment of the invention relates to methods of preparing modifiedcells for monitoring the level of a factor that is being administered toa subject for treatment for a disease or disorder, comprisingintroducing into cells of said subject (1) a polynucleotide encoding agene switch, said gene switch comprising at least one transcriptionfactor sequence, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, linked to a diagnosticswitch promoter, wherein the activity of the promoter is modulated bysaid factor that is being administered for treatment, and (2) apolynucleotide encoding a reporter gene linked to a promoter which isactivated by said ligand-dependent transcription factor, to producemodified cells.

Another embodiment of the invention relates to method of monitoring thelevel of a factor that is being administered to a subject for treatmentfor a disease or disorder, comprising:

-   (a) administering said treatment to said subject;-   (b) administering ligand to modified cells of said subject; and-   (c) detecting reporter gene expression;

wherein the level of expression of said reporter gene indicates thelevel of the factor being administered for treatment; and

wherein said modified cells of said subject comprise (1) apolynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, linked to a diagnostic switch promoter, wherein the activity ofthe promoter is modulated by said factor that is being administered fortreatment, and (2) a polynucleotide encoding a reporter gene linked to apromoter which is activated by said ligand-dependent transcriptionfactor.

In a further embodiment, the methods of monitoring the level of a factorthat is being administered to a subject for treatment for a disease ordisorder are carried out using non-autologous cells, e.g., cells thatare allogeneic or xenogeneic to the subject, and the modifiednon-autologous cells are introduced into the subject. In one embodiment,the non-autologous cells are surrounded by a barrier (e.g.,encapsulated) prior to being introduced into the subject. A method ofmonitoring the level of a factor that is being administered to a subjectfor a disease or disorder in a subject, comprising:

-   (a) introducing into non-autologous cells (1) a polynucleotide    encoding a gene switch, said gene switch comprising at least one    transcription factor sequence, wherein said at least one    transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated by said    factor that is being administered for treatment, and (2) a    polynucleotide encoding a reporter gene linked to a promoter which    is activated by said ligand-dependent transcription factor, to    produce modified cells;-   (b) introducing said modified cells into said subject;-   (c) administering ligand to said modified cells; and-   (d) detecting reporter gene expression;

wherein the level of expression of said reporter gene indicates thelevel of the factor being administered for treatment.

Another aspect of the invention relates to methods of detectingtransplant rejection in a subject that has received an organ or tissuetransplant, comprising:

-   (a) introducing into cells of said organ or tissue transplant (1) a    polynucleotide encoding a gene switch, said gene switch comprising    at least one transcription factor sequence, wherein said at least    one transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    transplant rejection, and (2) a polynucleotide encoding a reporter    gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression;

wherein expression of the reporter gene indicates that transplantrejection has been detected.

An additional embodiment of the invention relates to methods ofmonitoring the progression of transplant rejection in a subject that hasreceived an organ or tissue transplant, comprising:

-   (a) introducing into cells of said organ or tissue transplant (1) a    polynucleotide encoding a gene switch, said gene switch comprising    at least one transcription factor sequence, wherein said at least    one transcription factor sequence encodes a ligand-dependent    transcription factor, operably linked to a diagnostic switch    promoter, wherein the activity of the promoter is modulated during    transplant rejection, and (2) a polynucleotide encoding a reporter    gene linked to a promoter which is activated by said    ligand-dependent transcription factor, to produce modified cells;-   (b) administering ligand to said modified cells; and-   (c) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said transplant rejection in said subject.

The methods for detecting and monitoring transplant rejection in atransplant recipient can be used to monitor the viability oftransplanted organs or tissues. The ability to detect the onset oftransplant rejection will allow a medical practitioner to adjusttreatment of the transplant patient accordingly, e.g., by adjusting thelevel of immunosuppression therapy. The sensitivity of the presentmethods may allow for earlier detection of rejection than is possiblewith the current method of taking periodic tissue biopsies to look fortissue damage. The earlier detection of the occurrence of rejection in asubject will allow for a more rapid response and avoidance of furtherdamage to the transplanted organs or tissues. The methods may be usedfor any organ or tissue transplant, including, without limitation,heart, kidney, lung, liver, pancreas, small and large intestine, skin,cornea, bone marrow, bone, ligament, tendon, neural tissue, and stemcell transplants.

In the methods of the invention the gene switch comprises one or morediagnostic switch promoters that are activated during transplantrejection, i.e., rejection promoters. A rejection promoter is anypromoter that is activated in a transplanted organ or tissue when therejection process begins to occur. Examples of rejection promoters thatare useful in the present invention include, without limitation,promoters from the genes listed in Table 4, along with the organs inwhich increased expression has been detected during rejection.

TABLE 4 Gene Organ/Tissue A Disintegrin And Metalloproteinase 17(ADAM17) Kidney A Disintegrin And Metalloproteinase 19 (ADAM19) KidneyAGT Kidney Allograft Inflammatory Factor-1 (AIF-1) Liver Angiotensin IIType 1 Receptor (AGTR1) Heart APAF1 Intestine β2-Defensin Lung BrainNatriuretic Peptide Heart C-Reactive Protein (CRP) Liver C3 Kidney CCL1Heart CCL3 Heart CCL4 Heart CCL5 Heart CCR5 Heart CD3 Kidney CD95 LiverCD95 Ligand Liver, Heart CD103 Kidney Cellular Mediator of ImmuneResponse (MIR) Heart CFLAR Heart Chemokine (C—X—C motif) Ligand 9(CXCL9) Heart Collagen Type IX α3 Kidney Collagenase Lung ConnectiveTissue Growth Factor (CTGF) Kidney CX3CR1 Heart CXCR3 Pancreas, Heart,Kidney Cyclooxygenase-2 (COX-2) Kidney, Heart Early Growth ResponseGene-1 (EGR-1) Lung, Heart ENA 78 Heart Eotaxin Cornea Epidermal GrowthFactor Receptor (EGFR) Kidney EPST11 Intestine Fas Heart Fas LigandKidney, Heart, Pancreas Fork-Head Activin Signal Transducer-1 (FAST-1)Heart FOXP3 Kidney Fractalkine Kidney, Heart Gamma 2 Kidney GranulysinKidney Granzyme B Kidney, Pancreas, Intestine, Heart, Lung Heat ShockProtein-60 (HSP-60) Intestine Heat Shock Protein-70 (HSP-70) IntestineHepatocyte Growth Factor (HGF) Heart IF127 Intestine Integrin-α4 (ITGA4)Heart Interferon-γ Kidney, Intestine, Heart Interferon-Inducible Protein10 (IP-10; CXCL10) Kidney, Heart, Pancreas Interleukin-2 (IL-2) HeartInterleukin-2 Receptor (IL-2R) Intestine Interleukin-4 (IL-4) KidneyInterleukin-15 (IL-15) Heart, Lung Interleukin-18 (IL-18) KidneyIntracellular Adhesion Molecule-1 (ICAM-1) Kidney Laminin Kidney LAP3Intestine Macrophage Inflammatory Protein-2 (MIP-2) Cornea MADCAM-1Intestine Matrix Metalloproteinase-2 (MMP-2) Intestine, Kidney MatrixMetalloproteinase-9 (MMP-9) Kidney, Intestine MatrixMetalloproteinase-11 (MMP-11) Kidney Matrix Metalloproteinase-12(MMP-12) Kidney Matrix Metalloproteinase-14 (MMP-14) Kidney MDKIntestine MIG Pancreas, Heart MIP-1α Cornea, Pancreas, Heart, KidneyMIP-1β Cornea, Heart Monocyte Chemotactic Protein-1 (MCP-1) Cornea,Pancreas, Heart Monocyte Chemotactic Protein-2 (MCP-2) Heart MUC2Intestine MUC4 Intestine NKG2D Kidney p16 (INK4a) Kidney p21 (WAF/CIP1)Kidney p27 (Kip1) Kidney Perforin Kidney, Pancreas, Intestine, HeartProgrammed Cell Death (PDCD1) Heart RANTES Cornea, Pancreas, Kidney,Heart RAS Homolog Gene Family, Member U (RHOU) Heart Semaphorin 7A(SEMA7A) Heart Serine Proteinase Inhibitor-9 (PI-9) Kidney SOD2 HeartSTK6 Intestine Surfactant Protein-C (SP-C) Kidney TIAF-1 Kidney, LiverTIM-3 Kidney Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) KidneyTissue Inhibitor of Metalloproteinase-2 (TIMP-2) Kidney TransformingGrowth Factor-β1 (TGF-β1) Intestine, Kidney, Heart Transforming GrowthFactor Type I Receptor Intestine Tumor Necrosis Factor-α (TNF-α)Intestine, Heart, Kidney Urokinase Plasminogen Activator (uPA) KidneyUrokinase Plasminogen Activator Receptor (uPAR) Kidney Vascular CellAdhesion Molecule-1 (VCAM-1) Kidney, Lung Vasoactive Intestinal Peptide(VIP) Intestine WD Repeat Dommoain 40A (WDR40A) Heart XCL1 Heart

In one embodiment of the invention, the polynucleotides comprising therejection promoters are administered to the organ or tissue to betransplanted prior to the transplantation process. Organs and tissuesthat are used for transplantation typically must be transplanted into arecipient within 24-48 hours after removal from the donor. In oneembodiment, the polynucleotides of the invention are administered to theorgan or tissue within 48 hours of removal from the donor, e.g., within36, 24, 18, 12, or 6 hours of removal from the donor. In anotherembodiment, the polynucleotides are administered to the organ or tissueat least 48 hours prior to transplantation into the recipient, e.g., atleast 36, 24, 18, 12, or 6 hours prior to transplantation. Thepolynucleotides may be introduced into the organ or tissue to betransplanted in one location or in more than one location within theorgan or tissue, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more locations.

In another embodiment, the polynucleotides are administered to the organor tissue after it has been transplanted into a subject, e.g., 2, 4, 6,8, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeksor more after the transplantation.

The polynucleotides may be administered to the organ or tissue by anymeans as discussed above, including direct injection, electroporation,viral delivery, etc. In other embodiments, the polynucleotides may beadministered as part of transgenic cells, e.g., transgenic stem cells.The cells may be isolated from either the transplant donor or thetransplant recipient. For example, stem cells may be isolated from apatent in need of a transplant and the polynucleotides of the inventionintroduced into the stem cells. The transgenic cells may then be stored(e.g., frozen) until an organ or tissue is available fortransplantation. The transgenic cells may then be administered to theorgan or tissue before or after transplantation.

In a further embodiment, the methods of detecting transplant rejectionmay be carried out by introducing the polynucleotides of the inventioninto non-autologous cells, e.g., cells that are allogeneic or xenogeneicto the organ or tissue being transplanted, and the modifiednon-autologous cells are introduced to the organ or tissue prior totransplantation. In one embodiment, the modified non-autologous cellsare surrounded by a barrier (e.g., encapsulated) prior to beingintroduced into the organ or tissue.

In one embodiment of the invention, the gene switch comprises a singlerejection promoter operably linked to a transcription factor sequence.In another embodiment, the gene switch comprises two rejection promotersthat are operably linked to two different transcription factor sequencesthat together encode a ligand-dependent transcription factor. The tworejection promoters may be the same or different.

In another embodiment, the gene switch may further comprise a promoterthat regulates expression of a control protein that is useful formonitoring the function of the gene switch, i.e., to show that the geneswitch is operating properly in its environment, e.g., has not beensubjected to gene silencing. The expression of the control protein maybe used to limit false negative results from the diagnostic switch. Inone embodiment, the promoter linked to the control protein is aconstitutive promoter so that the control protein is always expressed.In a different embodiment, the promoter linked to the control protein isa switch promoter which is different from the rejection promoter(s)present in the gene switch. The control protein may be aligand-dependent transcription factor that binds to a promoter operablylinked to a second reporter gene that is different from the firstreporter gene. For example, the control protein may be aligand-dependent transcription factor having a different DNA bindingdomain than the transcription factor expressed from the rejectionpromoter(s), and that recognizes the response elements in the promoteroperably linked to the second reporter gene as shown in FIG. 4.

In one embodiment of the methods for detecting or monitoring transplantrejection, the reporter gene is any reporter gene described above. Inanother embodiment, the reporter gene encodes a secreted protein, e.g.,one that can be readily detected in a blood or urine sample of atransplant recipient. In another embodiment, the reporter gene encodes aprotein that is endogenous to the transplant recipient, e.g., a proteinthat is normally expressed at low levels so that an increase in reportergene expression upon the onset of rejection can be detected.

Once the polynucleotides of the invention have been administered to theorgan or tissue transplant, the methods of detecting or monitoringtransplant rejection may be carried out by detecting reporter geneexpression. In one embodiment, the level of reporter gene expression maybe measured once. In another embodiment, the level of reporter geneexpression may be measured more than once, e.g., regularly, such as onceevery 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4 weeks, or every 1, 2, 3, 4, 5, 6or more months. At each time point to be measured, a measurement ofreporter gene expression may be made in the absence of ligand to get abackground level of expression and in the presence ligand to obtain thelevel of reporter gene expression due to activation of the rejectionpromoter(s). In one embodiment, the level of reporter gene expression inthe presence of ligand is determined shortly after transplantation(e.g., within 1, 2, 3, 4, 5, 6 days or 1, 2, 3, or 4 weeks) to obtainthe ligand-induced baseline level of reporter gene expression prior tothe occurrence of any transplant rejection. The initial timepoint (orany subsequent timepoint) can be used to determine how much ligand mustbe administered to the subject and how long the ligand must be presentto obtain measurable reporter gene expression. Both the dose and timemay be adjusted as needed for each subject. Regular monitoring ofligand-induced reporter gene expression may then be carried out todetect any increase in reporter gene expression, which is indicative oftransplant rejection.

If a polynucleotide encoding a control protein (either constitutive orinducible) is present in the gene switch, the level of the controlprotein or the reporter gene induced by the control protein may bemeasured at the same time to confirm that the gene switch is functioningproperly. If the gene switch is not functioning optimally, it may benecessary to increase the ligand concentration or the amount of timebetween ligand administration and reporter gene detection to increasethe signal from the gene switch. In another embodiment, if the geneswitch is no longer functioning, additional polynucleotides may beadministered to the organ or tissue transplant so that monitoring oftransplant rejection can be continued.

In a further embodiment, once an increase in reporter gene expressionhas been detected indicating the presence of transplant rejection, thediagnosis may be confirmed using traditional means, e.g., by obtaining abiopsy of the transplanted tissue.

One embodiment of the invention relates to methods of preparing on organor tissue transplant for detecting transplant rejection in a subject,comprising introducing into cells of said organ or tissue transplant (1)a polynucleotide encoding a gene switch, said gene switch comprising atleast one transcription factor sequence, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, operably linked to a diagnostic switch promoter, wherein theactivity of the promoter is modulated during transplant rejection, and(2) a polynucleotide encoding a reporter gene linked to a promoter whichis activated by said ligand-dependent transcription factor, to producemodified cells.

Another embodiment of the invention relates to methods of detectingtransplant rejection in a subject that has received an organ or tissuetransplant, comprising:

-   (a) administering ligand to said subject; and-   (b) detecting reporter gene expression;

wherein expression of the reporter gene indicates that transplantrejection has been detected; and

wherein said organ or tissue transplant comprises one or more cellscomprising (1) a polynucleotide encoding a gene switch, said gene switchcomprising at least one transcription factor sequence, wherein said atleast one transcription factor sequence encodes a ligand-dependenttranscription factor, operably linked to a diagnostic switch promoter,wherein the activity of the promoter is modulated during transplantrejection, and (2) a polynucleotide encoding a reporter gene linked to apromoter which is activated by said ligand-dependent transcriptionfactor.

One embodiment of the invention relates to methods of preparing an organor tissue transplant for monitoring the progression of transplantrejection in a subject, comprising introducing into cells of said organor tissue transplant (1) a polynucleotide encoding a gene switch, saidgene switch comprising at least one transcription factor sequence,wherein said at least one transcription factor sequence encodes aligand-dependent transcription factor, operably linked to a diagnosticswitch promoter, wherein the activity of the promoter is modulatedduring transplant rejection, and (2) a polynucleotide encoding areporter gene linked to a promoter which is activated by saidligand-dependent transcription factor, to produce modified cells.

Another embodiment of the invention relates to methods of monitoring theprogression of transplant rejection in a subject that has received anorgan or tissue transplant, comprising:

-   (a) administering ligand to said subject; and-   (b) detecting reporter gene expression at least twice;

wherein a change in the level of expression of said reporter geneindicates progression of said transplant rejection in said subject, and

wherein said organ or tissue transplant comprises one or more cellscomprising (1) a polynucleotide encoding a gene switch, said gene switchcomprising at least one transcription factor sequence, wherein said atleast one transcription factor sequence encodes a ligand-dependenttranscription factor, operably linked to a diagnostic switch promoter,wherein the activity of the promoter is modulated during transplantrejection, and (2) a polynucleotide encoding a reporter gene linked to apromoter which is activated by said ligand-dependent transcriptionfactor.

In a further embodiment, the methods of monitoring transplant rejectionmay be carried out by introducing the polynucleotides of the inventioninto non-autologous cells, e.g., cells that are allogeneic or xenogeneicto the organ or tissue being transplanted, and the modifiednon-autologous cells are introduced to the organ or tissue prior totransplantation. In one embodiment, the modified non-autologous cellsare surrounded by a barrier (e.g., encapsulated) prior to beingintroduced into the organ or tissue.

In one embodiment of the methods described above, one or both of thepolynucleotides encoding a gene switch and a reporter gene may be partof a therapeutic vector that is being administered to a subject (e.g., avector encoding a therapeutic factor (protein or nucleic acid) for genetherapy). In this embodiment, the factor and the diagnostic test formonitoring the level of the factor are administered together in oneunit, ensuring that all cells that receive the treatment also receivethe diagnostic gene switch.

For each of the methods described above, in one embodiment, thepolynucleotide encoding the gene switch and the polynucleotide encodingthe reporter gene linked to a promoter are part of one largerpolynucleotide, e.g., a vector. In another embodiment, thepolynucleotide encoding the gene switch and the polynucleotide encodingthe reporter gene linked to a promoter are separate polynucleotides.

In one aspect, the invention relates to polynucleotides that may be usedin the methods of the invention. In one embodiment, the polynucleotideencodes a gene switch, the gene switch comprising at least onetranscription factor sequence, wherein said at least one transcriptionfactor sequence encodes a ligand-dependent transcription factor,operably linked to a diagnostic switch promoter, wherein the activity ofthe promoter is modulated during said disease or disorder. In anotherembodiment, the polynucleotide further encodes a reporter gene linked toa promoter which is activated by said ligand-dependent transcriptionfactor. In one embodiment, the gene switch is an EcR-based gene switch.In another embodiment, the gene switch comprises a first transcriptionfactor sequence under the control of a first diagnostic switch promoterand a second transcription factor sequence under the control of a seconddiagnostic switch promoter, wherein the proteins encoded by said firsttranscription factor sequence and said second transcription factorsequence interact to form a protein complex which functions as aligand-dependent transcription factor. In one embodiment, the firstdiagnostic switch promoter and the second diagnostic switch promoter aredifferent. In another embodiment, the first diagnostic switch promoterand the second diagnostic switch promoter are the same. In anotherembodiment, the first transcription factor sequence encodes a proteincomprising a heterodimer partner and a transactivation domain and thesecond transcription factor sequence encodes a protein comprising a DNAbinding domain and a ligand-binding domain. In a further embodiment, thepolynucleotide also encodes a lethal polypeptide operably linked to aninducible promoter.

In one aspect, the invention relates to polynucleotides encoding a geneswitch, the gene switch comprising at least one transcription factorsequence, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, linked to a diagnosticswitch promoter, wherein the activity of the promoter is modulated byfactors found in cells that are being exposed to toxic conditions. Inanother embodiment, the polynucleotide further encodes a reporter genelinked to a promoter which is activated by said ligand-dependenttranscription factor. In one embodiment, the gene switch is an EcR-basedgene switch. In another embodiment, the gene switch comprises a firsttranscription factor sequence under the control of a first diagnosticswitch promoter and a second transcription factor sequence under thecontrol of a second diagnostic switch promoter, wherein the proteinsencoded by said first transcription factor sequence and said secondtranscription factor sequence interact to form a protein complex whichfunctions as a ligand-dependent transcription factor. In one embodiment,the first diagnostic switch promoter and the second diagnostic switchpromoter are different. In another embodiment, the first diagnosticswitch promoter and the second diagnostic switch promoter are the same.In another embodiment, the first transcription factor sequence encodes aprotein comprising a heterodimer partner and a transactivation domainand the second transcription factor sequence encodes a proteincomprising a DNA binding domain and a ligand-binding domain. In afurther embodiment, the polynucleotide also encodes a lethal polypeptideoperably linked to an inducible promoter.

In one aspect, the invention relates to polynucleotides encoding a geneswitch, the gene switch comprising at least one transcription factorsequence, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, linked to a diagnosticswitch promoter, wherein the activity of the promoter is modulated bysaid factor that is being administered for treatment. In anotherembodiment, the polynucleotide further encodes a reporter gene linked toa promoter which is activated by said ligand-dependent transcriptionfactor. In one embodiment, the gene switch is an EcR-based gene switch.In another embodiment, the gene switch comprises a first transcriptionfactor sequence under the control of a first diagnostic switch promoterand a second transcription factor sequence under the control of a seconddiagnostic switch promoter, wherein the proteins encoded by said firsttranscription factor sequence and said second transcription factorsequence interact to form a protein complex which functions as aligand-dependent transcription factor. In one embodiment, the firstdiagnostic switch promoter and the second diagnostic switch promoter aredifferent. In another embodiment, the first diagnostic switch promoterand the second diagnostic switch promoter are the same. In anotherembodiment, the first transcription factor sequence encodes a proteincomprising a heterodimer partner and a transactivation domain and thesecond transcription factor sequence encodes a protein comprising a DNAbinding domain and a ligand-binding domain. In a further embodiment, thepolynucleotide also encodes a lethal polypeptide operably linked to aninducible promoter.

In one aspect, the invention relates to polynucleotides encoding a geneswitch, the gene switch comprising at least one transcription factorsequence, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, linked to a diagnosticswitch promoter, wherein the activity of the promoter is modulatedduring transplant rejection. In another embodiment, the polynucleotidefurther encodes a reporter gene linked to a promoter which is activatedby said ligand-dependent transcription factor. In one embodiment, thegene switch is an EcR-based gene switch. In another embodiment, the geneswitch comprises a first transcription factor sequence under the controlof a first diagnostic switch promoter and a second transcription factorsequence under the control of a second diagnostic switch promoter,wherein the proteins encoded by said first transcription factor sequenceand said second transcription factor sequence interact to form a proteincomplex which functions as a ligand-dependent transcription factor. Inone embodiment, the first diagnostic switch promoter and the seconddiagnostic switch promoter are different. In another embodiment, thefirst diagnostic switch promoter and the second diagnostic switchpromoter are the same. In another embodiment, the first transcriptionfactor sequence encodes a protein comprising a heterodimer partner and atransactivation domain and the second transcription factor sequenceencodes a protein comprising a DNA binding domain and a ligand-bindingdomain. In a further embodiment, the polynucleotide also encodes alethal polypeptide operably linked to an inducible promoter.

Another aspect of the invention relates to vectors comprising any of thepolynucleotides described above. In one embodiment, the vector is aplasmid vector or a viral vector. In one embodiment, the polynucleotidesare present on the same vector. In a further embodiment, each of thepolynucleotides is on a separate vector. The separate vectors may be theidentical vector (e.g., the same plasmid), the same type of vector(e.g., both are plasmids but not the same plasmid), or different typesof vectors (e.g., one vector is a plasmid, the other vector is a virus).

In another aspect, the invention provides kits that may be used inconjunction with methods the invention. Kits according to this aspect ofthe invention may comprise one or more containers, which may contain oneor more components selected from the group consisting of one or morenucleic acid molecules, restriction enzymes and one or more cellscomprising such nucleic acid molecules. Kits of the invention mayfurther comprise one or more containers containing cell culture mediasuitable for culturing cells of the invention, one or more containerscontaining antibiotics suitable for use in culturing cells of theinvention, one or more containers containing buffers, one or morecontainers containing transfection reagents, one or more containerscontaining substrates for enzymatic reactions, and/or one or moreligands for gene switch activation.

Kits of the invention may contain a wide variety of nucleic acidmolecules that can be used with the invention. Examples of nucleic acidmolecules that can be supplied in kits of the invention include thosethat contain promoters, sequences encoding gene switches, enhancers,repressors, selection markers, transcription signals, translationsignals, primer hybridization sites (e.g., for sequencing or PCR),recombination sites, restriction sites and polylinkers, sites thatsuppress the termination of translation in the presence of a suppressortRNA, suppressor tRNA coding sequences, sequences that encode domainsand/or regions, origins of replication, telomeres, centromeres, and thelike. In one embodiment, kits may comprise a polynucleotide comprising agene switch without any diagnostic switch promoters, the polynucleotidebeing suitable for insertion of any diagnostic switch promoters ofinterest. Nucleic acid molecules of the invention may comprise any oneor more of these features in addition to polynucleotides as describedabove.

Kits of the invention may comprise cells. The cells may comprise thepolynucleotides of the invention, or the cells and the polynucleotidesmay be in separate containers. In one embodiment the cells may beautologous cells, e.g., as part of a kit designed for a specificsubject. In another embodiment, the cells may be non-autologous cells,e.g., as part of a kit designed for any subject. In a furtherembodiment, the non-autologous cells may be surrounded by a barrier(e.g., encapsulated).

Kits of the invention may comprise containers containing one or morerecombination proteins. Suitable recombination proteins include, but arenot limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3resolvase, ΦC31, TndX, XerC, and XerD. Other suitable recombinationsites and proteins are those associated with the GATEWAY™ CloningTechnology available from Invitrogen Corp., Carlsbad, Calif., anddescribed in the product literature of the GATEWAY™ Cloning Technology(version E, Sep. 22, 2003), the entire disclosures of which areincorporated herein by reference.

Kits of the invention can also be supplied with primers. These primerswill generally be designed to anneal to molecules having specificnucleotide sequences. For example, these primers can be designed for usein PCR to amplify a particular nucleic acid molecule. Sequencing primersmay also be supplied with the kit.

One or more buffers (e.g., one, two, three, four, five, eight, ten,fifteen) may be supplied in kits of the invention. These buffers may besupplied at working concentrations or may be supplied in concentratedform and then diluted to the working concentrations. These buffers willoften contain salt, metal ions, co-factors, metal ion chelating agents,etc. for the enhancement of activities or the stabilization of eitherthe buffer itself or molecules in the buffer. Further, these buffers maybe supplied in dried or aqueous forms. When buffers are supplied in adried form, they will generally be dissolved in water prior to use.

Kits of the invention may contain virtually any combination of thecomponents set out above or described elsewhere herein. As one skilledin the art would recognize, the components supplied with kits of theinvention will vary with the intended use for the kits. Thus, kits maybe designed to perform various functions set out in this application andthe components of such kits will vary accordingly.

The present invention further relates to instructions for performing oneor more methods of the invention. Such instructions can instruct a userof conditions suitable for performing methods of the invention.Instructions of the invention can be in a tangible form, for example,written instructions (e.g., typed on paper), or can be in an intangibleform, for example, accessible via a computer disk or over the internet.

It will be recognized that a full text of instructions for performing amethod of the invention or, where the instructions are included with akit, for using the kit, need not be provided. One example of a situationin which a kit of the invention, for example, would not contain suchfull length instructions is where the provided directions inform a userof the kits where to obtain instructions for practicing methods forwhich the kit can be used. Thus, instructions for performing methods ofthe invention can be obtained from internet web pages, separately soldor distributed manuals or other product literature, etc. The inventionthus includes kits that direct a kit user to one or more locations whereinstructions not directly packaged and/or distributed with the kits canbe found. Such instructions can be in any form including, but notlimited to, electronic or printed forms.

Having now fully described the invention, it will be understood by thoseof ordinary skill in the art that the same can be performed within awide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

EXAMPLES Example 1

The vector shown in FIG. 5 includes IL-24/mda-7 promoter (SEQ ID NO.:5). Adenovirus produced using the adenoviral shuttle vector is used totransduce cells isolated from lymphatic samples. The transduces cellsare split into two groups and cultured either in the presence or absenceof activator ligand. Both sets of cells are then disrupted, and theresulting cell lysates are used in luciferase assays.

Example 2

The vector shown in FIG. 6 includes TRPM4 and TRGC1/TARP promoters (SEQID NO.: 6). The DNA vector is used to transduce a prostate biopsy usingnon-viral transduction systems. The transduced biopsy is split into twogroups and cultured either in the presence or absence of an activatorligand. Both sets of biopsied tissues are homogenized, and the resultinglysates are used in luciferase assays.

Example 3

The vector shown in FIG. 7 includes the ADAM-17 promoter (SEQ ID NO.: 7)and the CD95-ADAM8 reporter (SEQ ID NOs: 10-11). Adenovirus producedusing the adenoviral shuttle vector is used to transduced a portion of adonor kidney before transplantation. Following transplantation, serumsamples are assayed for the reporter gene for a period defined by thephysician. The assay protocol consists of assaying reporter expressionfrom samples not exposed to activator ligand. Immediately after blooddraw of the non-activated group, ligand is administered for 24 hours andthen discontinued; another blood sample is acquired within one hourbefore the ligand treatment is completed. Reporter assay results arecompared between the ligand-treated and untreated samples. A period of60 hours must pass before this procedure is performed again.

Example 4

The vector shown in FIG. 8 includes the CXCL9 and SEMA7A promoters (SEQID NO.: 8) and the CD40-CD3 reporter (SEQ ID NOs.: 12-13). This vectoris used to transduce a portion of a donor heart before transplantationvia direct needle injection. Following transplantation, serum samplesare assayed for the reporter gene for a period defined by the physician.The assay protocol consists of assaying reporter expression from samplesnot exposed to an activator ligand. Immediately after the blood draw ofthe “non-activated” group, ligand is administered for 24 hours and thendiscontinued; another blood sample is acquired within one hour beforethe ligand treatment is completed. Reporter assay results are comparedbetween the ligand-treated and untreated samples. A period of 60 hoursmust pass before this procedure can be performed again.

Example 5

The vector shown in FIG. 9 includes the ADAM-17 promoter and thealkaline phosphatase-c terminal CD40 reporter (SEQ ID NO.: 14).Adenovirus produced using this adenoviral shuttle vector is used totransduce primary porcine kidney cells. The resulting cells areencapsulated in alginate (see WO 2007/046719A2), and then implanted intoa donor kidney before transplantation. Following transplantation, serumsamples are assayed for the reporter gene for a period defined by thephysician. The assay protocol consists of assaying reporter expressionfrom samples not exposed to activator ligand. Immediately after theblood draw of the “non-activated” group, ligand is administered for 24hours and then discontinued; another blood sample is acquired within onehour before the ligand treatment is completed. Reporter assay resultsare compared between the ligand-treated and untreated samples. A periodof 60 hours must pass before this procedure can be performed again.

1-38. (canceled)
 39. A method of monitoring the effectiveness of atreatment for a disease or disorder in a subject, comprising: (a)administering said treatment to said subject; (b) introducing into cellsof said subject a polynucleotide, wherein said polynucleotide encodes agene switch, wherein said polynucleotide comprises (1) at least onetranscription factor sequence, which is operably linked to a diagnosticswitch promoter, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, wherein the activity ofsaid promoter is modulated during said disease or disorder of saidsubject, and (2) a polynucleotide encoding a reporter gene linked to apromoter which is activated by said ligand-dependent transcriptionfactor, to produce modified cells; (c) administering ligand to saidmodified cells; and (d) detecting reporter gene expression at leasttwice; wherein a change in the level of expression of said reporter geneindicates the effectiveness of said treatment.
 40. The method of claim39, wherein step (b) is carried out before step (a).
 41. The method ofclaim 40, wherein a baseline level of reporter gene expression isdetermined prior to step (a).
 42. The method of claim 39, wherein saidmethod is carried out ex vivo in cells that have been isolated from saidsubject.
 43. The method of claim 39, wherein said polynucleotides areintroduced into cells that have been isolated from said subject toproduce modified cells, and the modified cells are re-introduced intosaid subject.
 44. The method of claim 39, wherein said method is carriedout in vivo.
 45. The method of claim 39, wherein said gene switch is anEcR-based gene switch.
 46. The method of claim 45, wherein said ligandbinds to the EcR ligand binding domain.
 47. The method of claim 46,wherein said ligand is a diacylhydrazine.
 48. The method of claim 47,wherein said ligand is selected from the group consisting of RG-115819,RG-115830, and RG-115932.
 49. The method of claim 46, wherein saidligand is an amidoketone or oxadiazoline.
 50. The method of claim 39,wherein said gene switch comprises a first transcription factor sequenceunder the control of a first diagnostic switch promoter and a secondtranscription factor sequence under the control of a second diagnosticswitch promoter, wherein the proteins encoded by said firsttranscription factor sequence and said second transcription factorsequence interact to form a protein complex which functions as aligand-dependent transcription factor.
 51. The method of claim 50,wherein said first diagnostic switch promoter and said second diagnosticswitch promoter are different.
 52. The method of claim 50, wherein saidfirst diagnostic switch promoter and said second diagnostic switchpromoter are the same.
 53. The method of claim 50, wherein said firsttranscription factor sequence encodes a protein comprising a heterodimerpartner and a transactivation domain.
 54. The method of claim 50,wherein said second transcription factor sequence encodes a proteincomprising a DNA binding domain and a ligand-binding domain.
 55. Themethod of claim 39, wherein one of said polynucleotides further encodesa lethal polypeptide operably linked to an inducible promoter. 56-59.(canceled)
 60. A method of monitoring the potential toxicity of anadministered treatment for a disease or disorder in a subject,comprising: (a) administering said treatment to said subject; (b)introducing into cells of said subject a polynucleotide, wherein saidpolynucleotide encodes a gene switch, wherein said polynucleotidecomprises (1) at least one transcription factor sequence, which isoperably linked to a diagnostic switch promoter, wherein said at leastone transcription factor sequence encodes a ligand-dependenttranscription factor, wherein the activity of said promoter is modulatedby factors found in cells that are being exposed to toxic conditions,and (2) a polynucleotide encoding a reporter gene linked to a promoterwhich is activated by said ligand-dependent transcription factor, toproduce modified cells; (c) administering ligand to said modified cells;and (d) detecting reporter gene expression at least twice; wherein achange in the level of expression of said reporter gene indicates thetoxicity of said treatment. 61-80. (canceled)
 81. A method of monitoringthe level of a factor that is being administered to a subject fortreatment for a disease or disorder, comprising: (a) administering saidtreatment to said subject; (b) introducing into cells of said subject apolynucleotide, wherein said polynucleotide encodes a gene switch,wherein said polynucleotide comprises (1) at least one transcriptionfactor sequence, which is operably linked to a diagnostic switchpromoter, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, wherein the activity ofsaid promoter is modulated by said factor that is being administered fortreatment, and (2) a polynucleotide encoding a reporter gene linked to apromoter which is activated by said ligand-dependent transcriptionfactor, to produce modified cells; (c) administering ligand to saidmodified cells; and (d) detecting reporter gene expression; wherein thelevel of expression of said reporter gene indicates the level of thefactor being administered for treatment. 82-102. (canceled)
 103. Amethod of detecting transplant rejection in a subject that has receivedan organ or tissue transplant, comprising: (a) introducing into cells ofsaid organ or tissue transplant a polynucleotide, wherein saidpolynucleotide encodes a gene switch, wherein said polynucleotidecomprises (1) at least one transcription factor sequence, which isoperably linked to a diagnostic switch promoter, wherein said at leastone transcription factor sequence encodes a ligand-dependenttranscription factor, wherein the activity of said promoter is modulatedduring transplant rejection, and (2) a polynucleotide encoding areporter gene linked to a promoter which is activated by saidligand-dependent transcription factor, to produce modified cells; (b)administering ligand to said modified cells; and (c) detecting reportergene expression; wherein expression of the reporter gene indicates thattransplant rejection has been detected. 104-175. (canceled)